CN107028886A - Lipid particle and related methods containing nucleic acid - Google Patents

Lipid particle and related methods containing nucleic acid Download PDF

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Publication number
CN107028886A
CN107028886A CN201710181826.6A CN201710181826A CN107028886A CN 107028886 A CN107028886 A CN 107028886A CN 201710181826 A CN201710181826 A CN 201710181826A CN 107028886 A CN107028886 A CN 107028886A
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lipid
particle
lnp
sirna
nucleic acid
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P·R·库里斯
N·M·贝利维奥
C·L·G·汉森
J·胡弗特
J·泰勒
A·维尔德
S·马尔克姆
I·哈弗兹
A·勒恩格
D·沃克
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University of British Columbia
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University of British Columbia
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/145—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Abstract

The present invention provides the method using nucleic acid lipid nanoparticle of the microfluid mixing device preparation comprising cation lipid DLinKC2 DMA, wherein obtaining nucleic acid lipid nanoparticle presentation smaller particle and bigger core density that nucleic acid lipid nanoparticle compares the identical preparation produced in the vesica method routinely carried out.

Description

Lipid particle and related methods containing nucleic acid
It is on November 4th, 2010, the division of the application of the same name of Application No. 201080059999.7 applying date that the application, which is, Application.
【Technical field】
The U.S. Provisional Application No.61/280 submitted this application claims on November 4th, 2009,510 rights and interests are passed through Reference is integrally incorporated herein.
【Background technology】
Lipid nanoparticle (LNP) be have received regulation and control approval most clinical progress have the medicine based on 7 LNP Drug delivery system.The medicine of these approvals contains small molecule such as cancer therapy drug and is presented what is improved compared to " dissociating " medicine Effect and/or the toxicity of reduction.LNP carrier techniques have also been applied to delivering " heredity " medicine such as express therapeutic albumen Or the plasmid of siRNA (siRNA) oligonucleotides (OGN) of the gene of progression of disease is contributed to for silence.siRNA OGN The method made from effective delivering in vivo of other genetic medicines is the revolutionary potentiality for hindering these agent as therapeutic agent Subject matter.
The design of the newly-developed and cation lipid of the LNP technologies that the parcel of genetic medicine and delivering need is lighted LNP systems solve the problems, such as the potentiality of delivering in vivo.LNP-siRNA systems are induced after being shown in intravenous (intravenous) injection The silence of the target gene of association is treated in animal model, including non-human primate, and is commented at present in several clinical tests Under estimating.
Various methods have been developed to prepare the LNP systems containing genetic medicine.These methods are included in the presence of ethanol The pre-formed LNP of mixing and the lipid of OGN or mixed dissolution in ethanol and the aqueous medium containing OGN and obtain with 100nm Or the LNP and 65~95% of less diameter OGN parcel efficiency.These methods all rely on the presence of cation lipid, with The parcel of OGN and PEG (PEG) lipid is reached, to suppress the aggregation and formation of big structure.The property of the LNP systems of generation Matter, including size and OGN parcel efficiency, are sensitive to various formulation parameters such as ionic strength, lipid and concentration of alcohol, pH, OGN Concentration and mixing velocity.In general, using present agent process be difficult to control parameter such as mixing when relative lipid with OGN concentration, and mixing velocity, cause within prepared product and between the variability of LNP feature that produces.
Microfluidic device is provided with nl yardsticks with controlling and the ability of quick fluid-mixing, precise control of temperature, is resident Time and solute concentration.Control and quick Microfluidic Mixing is applied to synthesis inorganic nano-particle and particulate before, and can Surpass the extensive aborning large scale system of nano-particle.Microfluid 2- phases drip technology and have been applied to produce monodisperse polymer Thing particulate is used for medicine delivery or for producing the big vesica for cell encapsulation, albumen or other biological molecule.Have shown that water Kinetic current is focused on, and the use of common micro-fluidic technologies is to provide the quick mixing of reagent, with single point of the size that creates control Dissipate liposome.This technology is also proved to compare body production method, for the higher parcel of small molecule, obtaining smaller, more The generation of the polymer nano-particle of many monodisperse particles is useful.
Although the development of the exploitation of the method for the LNP systems containing genetic medicine, exist and therapeutic substance is contained to preparation Lipid nanoparticle apparatus and method, and improve the lipid nanoparticle containing therapeutic substance demand.This hair It is bright to seek to realize this demand and further related advantage is provided.
【Summary of the invention】
On the one hand, the present invention provides the lipid particle for including nucleic acid.
In one embodiment, lipid particle includes (a) one or more of cation lipids, the 2nd fat of (b) one or more Matter, and (c) one or more of nucleic acid, wherein lipid particle include substantially entity core, as defined herein.
In one embodiment, cation lipid is DLin-KC2-DMA.In certain embodiments, particle includes about 30 ~about 95 molar percentage cation lipids.
In one embodiment, the 2nd lipid is PEG-c-DMA.In one embodiment, the 2nd lipid is 1,2- distearyls Acyl-sn- glyceryl -3- phosphocholines (DSPC).In certain embodiments, particle includes about 1~about 10 molar percentage the 2nd Lipid.
Nucleic acid can be DNA, RNA, lock nucleic acid, nucleic acid analog, or can express DNA or RNA plasmid.
In another embodiment, lipid particle includes (a) one or more of cation lipids, during (b) is one or more of Property lipid, (c) one or more PEG- lipids, (d) one or more sterols；And (e) one or more of nucleic acid, wherein liposome Attached bag contains substantially entity core, as defined herein.In one embodiment, cation lipid is DLin-KC2-DMA.One In embodiment, PEG- lipids are PEG-c-DMA.In one embodiment, neutral lipid is 1,2- distearyl-sn- glycerine Base -3- phosphocholines (DSPC).In one embodiment, sterol is cholesterine.In one embodiment, nucleic acid is siRNA.
In further embodiment, lipid particle is by one or more of cation lipids, and one or more of nucleic acid groups Into.In one embodiment, lipid particle includes substantially entity core, as defined herein.In one embodiment, cation Lipid is DLin-KC2-DMA.In one embodiment, nucleic acid is siRNA.
In other respects, the present invention provides the method using lipid particle.
In one embodiment, the method that the present invention is supplied to subject's administration of nucleic acid, including apply the lipid of the present invention Particle gives the subject for having demand.
In one embodiment, the present invention is provided the method for nucleic acid into cells, including makes the cells contacting present invention's Lipid particle.
In one embodiment, the present invention provides the method for the expression of regulation target polynucleotide or polypeptide, including makes cell The lipid particle of the present invention is contacted, its amplifying nucleic acid can adjust the expression of target polynucleotide or polypeptide.
In one embodiment, the present invention provides the disease or illness for the overexpression that polypeptide is characterized as in treatment subject Method, including the lipid particle to the subject administration present invention, its amplifying nucleic acid energy silence or reduction expression of polypeptides.
In other respects, the method that the present invention provides manufacture lipid particle.
In one embodiment, the method that the present invention provides lipid particle of the manufacture containing nucleic acid, including：
(a) the 1st conductance for including nucleic acid in the 1st solvent is entered into microfluidic device；Wherein device, which has, is suitable to one Or more the 1st area of conductance inflow device and for the 2nd area of the content that one or more streams are mixed with microfluid mixer；
(b) the 2nd comprising lipid particle-forming material in the 2nd solvent is flowed gatherer to provide in laminar flow condition 1st and the 2nd stream of lower flowing, wherein device, which have, to be suitable to one or more conductances flowing into the 1st area of microchannel and for mixing The 2nd area of the content of one or more streams is closed, wherein lipid particle-forming material includes cation lipid, and wherein the 1st He 2nd solvent is differed；
(c) one or more 1st streams and one or more 2 streams are made from the 2nd area of the 1st area's inflow device of device；With And
(d) one or more 1 streams and one or more the flowed under laminar flow condition are blended in the 2nd area of device The content of 2 streams flows with the provide the lipid nanoparticle for including the nucleic acid with parcel the 3rd.
In another embodiment, the method that the present invention provides lipid particle of the manufacture containing nucleic acid, including：
(a) by the 1st stream introduction channel comprising nucleic acid in the 1st solvent；Wherein device have be suitable to will be one or more 1st area of conductance flow channel and for the 2nd area of the content for mixing one or more streams；
(b) the 2nd stream comprising lipid particle-forming material in the 2nd solvent is imported；Wherein passage, which has, is suitable to one Or more the 1st area of conductance flow channel and for the 2nd area of the content for mixing one or more streams；
(c) one or more 1st streams and one or more 2 streams are made from the 2nd area of the 1st area's flow channel of passage, and The physical isolation of 2 stream is maintained, the stream of one of them or more the 1st and one or more 2 streams are not mixed first, until reaching passage The 2nd area after just mix；And
(d) one or more 1 flowed under laminar flow condition are blended in the 2nd area of microchannel to flow and one or more The content of 2nd stream flows with the provide the lipid nanoparticle for including the nucleic acid with parcel the 3rd.
In the particular implementation of above method, the content of one or more 1st streams and one or more 2 streams is mixed Thing includes changing the concentration or relative mixing velocity of one or more 1st streams and one or more 2 streams.
In the particular implementation of above method, method is also included to be flowed with aqueous buffered dilution agent the 3rd.In particular implementation In mode, the stream of dilution the 3rd, which is included, makes the 3rd stream and aqueous buffer flow into the 2nd mixed structure.
In the particular implementation of above method, method also contains the lipid particle of the nucleic acid with parcel comprising dialysis bag Aqueous buffer to reduce the amount of the 2nd solvent.
In the particular implementation of above method, the 1st solvent is aqueous buffer.In the particular implementation side of above method In formula, the 2nd solvent is aqueous alcohol.
In the particular implementation of above method, the content of the stream of mixing the 1st and the 2nd includes mixed and disorderly advection. In the particular implementation of above method, the content of the stream of mixing the 1st and the 2nd includes being mixed with micro-mixer.
In the particular implementation of above method, nucleic acid parcel efficiency is about 90~about 100%.
In the particular implementation of above method, one or more 1 streams and one or more 2 streams are blended in the Prevented in 1st area by barrier.In certain embodiments, barrier is conduit wall, sheath fluid or concentric pipe.
There is provided the device of manufacture lipid particle in another aspect of this invention.In one embodiment, the present invention provides production The device of the lipid particle of raw parcel nucleic acid, including：
(a) the 1st entrance, it is used to receive the 1st solution for including nucleic acid in the 1st solvent；
(b) the 1st inlet microchannel, it circulates with the 1st entrance is connected to provide the 1st stream comprising nucleic acid in the 1st solvent；
(c) the 2nd entrance, it is used to receive the 2nd solution for including lipid particle-forming material in the 2nd solvent；
(d) the 2nd inlet microchannel, it is connected with the circulation of the 2nd entrance and lipid particle-shape is included in the 2nd solvent to provide Into the 2nd stream of material；And
(e) the 3rd microchannel, its be used for receive the 1st and the 2nd flow, wherein the 3rd microchannel have be suitable under laminar flow condition will 1st and the 2nd conductance flows into the 1st area of microchannel and the 2nd area of the content flowed suitable for mixing the 1st and the 2nd and includes tool to provide There is the 3rd stream of the lipid particle of the nucleic acid of parcel.
In one embodiment, device also includes stabilized to provide including for dilution for diluting the facility of the 3rd stream The stream of the lipid particle of nucleic acid with parcel.In certain embodiments, microring array is included for diluting the facility of the 3rd stream Device.
In one embodiment, microchannel has about 20~about 300 μm of hydrodynamic diameter.
In one embodiment, the 2nd area of microchannel includes basse-taille structure.In one embodiment, the 2nd of microchannel Area has main flow direction and one or more surfaces, and there is at least one to be defined in groove therein or projection on the surface, recessed Groove or projection have the orientation with principal direction angulation.In one embodiment, the 2nd area includes micro-mixer.
In certain embodiments, device also includes the facility for the flow velocity for being used to change the 1st and the 2nd stream.
In certain embodiments, device is included also in the 1st area for the stream of physical isolation the one or more 1st and one Or more the 2nd effective barrier of stream.
【Brief description of the drawings】
Aforementioned aspect of the present invention and it is many with advantage can become easier to be agreed for by reference to as detailed below, The identical more preferably understood referring to the drawings.
Fig. 1 is the schematic diagram of the representative fluid means of the present invention.
Fig. 2 is the schematic diagram of the representative fluid means of the present invention, and it is the detail of the device of illustration in Fig. 1.
Fig. 3 is the schematic diagram of the representative fluid means of the present invention, and it is the detail of the device of illustration in Fig. 2.
Fig. 4 is the representative fluid means of the present invention and the schematic diagram of method.
Fig. 5 is the schematic diagram of the representative array of the present invention, and it includes 10 in the fluid means of illustration in Fig. 4.
Fig. 6 is the schematic diagram of the representative fluid means of the present invention.
Fig. 7 is the schematic diagram of the representative array of the present invention, and it includes 10 in the representative fluid means of illustration in Fig. 6.
Fig. 8 is the schematic diagram of the representative fluid means of the present invention, and it has 3 entrances and single outlet, and (device 800 includes Hybrid channel 810).
Fig. 9 is the schematic diagram of the representative fluid means of the present invention, and it has 2 entrances and single outlet, and (device 900 includes Hybrid channel 910).
Figure 10 is the schematic diagram of the representative fluid means of the present invention, and it has multiple (n) series entrance and single outlet (device 1000 includes hybrid channel 1010a, 1010b, 1010c and 1010d).
Figure 11 is the schematic diagram of the representative fluid means of the present invention, and it has 3 entrances and single outlet, and (device 1100 is wrapped Include hybrid channel 1110a, 1110b and 1110c).
Figure 12 is the schematic diagram of the representative fluid means of the present invention, and it has 7 entrances and single outlet, and (device 1200 is wrapped Include hybrid channel 1210a, 1210b, 1210c and 1210d).
Figure 13 is the schematic diagram of the representative fluid means of the present invention, and it has multilayer blender, and (device 1300 includes mixing Passage 1310).
Figure 14 is the close up view of the multilayer blender of illustration in Figure 14.
Figure 15 A are the schematic diagrames of representative microfluid (MF) method of the invention for manufacturing lipid nanoparticle (LNP)：Fat Matter-ethanol and the siRNA- aqueous solution are pumped into the entrance of microfluid mixing device；The herringbone feature of device induces the miscellaneous of laminar stream Random advection and lipid matter is caused quickly to mix and formed lipid nanoparticle with current.Hybrid channel is 200 μm wide and 79 μm It is high.Herringbone structure is 31 μm high and 50 μ m-thicks.
Figure 15 B are the schematic diagrames of pre-formed vesica (PFV) method for manufacturing lipid nanoparticle (LNP)：(a) by fat Matter-ethanol solution adds the aqueous solution, and pH4.0 causes the formation of vesica particles of types；(b) 80nm polycarbonate membranes are passed through (Nuclepore) more homogeneous particle is provided using the extrusion of Lipex extruders in room temperature to be distributed；And (c) addition siRNA solution And the parcel for promoting siRNA for 30 minutes that is vortexed and incubated in 35 DEG C.
Flow velocity in Figure 16 A~16C illustration microfluidic devices is to mixing and the influence of LNP particle diameters.2 10 μM of fluoresceins (in pH8.8 fluorescence, in pH5.15 non-fluorescence) solution is blended to produce complete fluorescent solutions.Figure 16 A are compared by along logical The mixability (%) that the average fluorescent strength of road width is determined as from mean fluid velocity and length of travel (0.2,0.8, 1.4 and 2mL/min) calculate incorporation time (msec) function.Figure 16 B and 16C compare by with 40:11.5:47.5:1 rubs You compare, and the LNP's of Dlin-KC2-DMA/DSPC/ cholesterine of the siRNA- total lipids than 0.06wt/wt/PEG-c-DMA compositions is flat Equal particle diameter, 10mM lipids-ethanol phase and the 25mM acetate buffers containing siRNA, pH4 mixing.Figure 16 B compare the flat of LNP Equal particle diameter (nm) as flow velocity (mL/min) function.Figure 16 C compare LNP average grain diameter (nm) as ethanol/water velocity ratio Function.Error bars represent the standard deviation of the average grain diameter by dynamic light scattering measurement.
Figure 17 is by comparing average grain diameter (nm) as the function illustration lipid concentration pair of the lipid concentration (mM) in ethanol The influence of LNP particle diameters.Increase lipid concentration causes average grain diameter to increase.Total fat in the ethanol phase mixed in micro-fluid chip It is 10mM~50mM that matter content, which changes,.LNP is by with 40:11.5:47.5:1 mol ratio, siRNA- total lipids compare 0.06wt/wt Dlin-KC2-DMA/DSPC/ cholesterine/PEG-c-DMA composition.Overall flow rate within microfluid mixer maintains 2ml/ min.Error bars represent the standard deviation of the average grain diameter by dynamic light scattering measurement.
The influence of Figure 18 A and 18B illustration PEG- lipids and cationic lipid confrontation LNP systems.Figure 18 A compare average grain diameter (nm) as the function for the PEG-c-DMA contents (mol% in LNP) for passing through LNP prepared by PFV and MF methods.PEG- lipids Changed in LNP compositions is 1mol%~10mol%.The change of PEG- lipid contents is mended by adjusting cholesterol content Repay.LNP is by with 40:11.5:47.5:1(-x):1 (+x) mol ratio, (wherein x=1~9), siRNA- total lipids ratio 0.06wt/wt Dlin-KC2-DMA/DSPC/ cholesterine/PEG-c-DMA compositions.Figure 18 B compare average grain diameter (nm) as logical Cross the function of the LNP of PFV and MF methods preparation DLin-KC2-DMA contents (mol%).It is 40mol% that cation lipid, which changes, ~70mol%.PEG-c-DMA is held constant at 1mol% and maintains 0.25 mol ratio with DSPC- cholesterine.Microfluid mixer Within overall flow rate maintain 2ml/min.10mM lipids-ethanol phase and the 25mM acetate buffers containing siRNA, pH4 are mixed Close.Error bars represent the standard deviation of the average grain diameter by dynamic light scattering measurement.
Figure 19 is used as siRNA/ lipids ratio (wt/wt) and (is also expressed as core by comparing average grain diameter (nm) and parcel (%) Thuja acid/phosphoric acid (N/P)) function and illustration siRNA/ lipids compare particle diameter and parcel influence.By using anion exchange Revolve post and determine parcel from free siRNA separation LNP suspension.LNP is by with 40:11.5:47.5:1 mol ratio, siRNA- is total Dlin-KC2-DMA/DSPC/ cholesterine of the lipid than 0.06wt/wt/PEG-c-DMA compositions.It is total within microfluid mixer Flow velocity maintains 2ml/min.10mM lipids-ethanol phase and the 25mM acetate buffers containing siRNA, pH4 mixing.Error bars Represent the standard deviation of the average grain diameter by dynamic light scattering measurement.
Figure 20 A and 20B illustration are prepared by microfluid mixer using freezing-transmission Electron microscopy (TEM) The form of PEG- lipids and cation lipid LNP systems.LNP is imaged by Cryo-TEM with 29K magnifying powers.Figure 20 A be by with 40:11.5:47.5:The empty LNP of the Dlin-KC2-DMA/DSPC/ cholesterine of 1 mol ratio/PEG-c-DMA compositions picture.Figure 20B is by with 40:11.5:47.5:1 mol ratio, Dlin-KC2-DMA/DSPC/ courage of the siRNA- total lipids than 0.06wt/wt The picture for the LNP that the siRNA of sterol/PEG-c-DMA compositions is loaded.Entered using microfluid mixer with 20mM lipids in ethanol phase Row preparation.Same form is presented in the LNP-siRNA and empty particle of loading containing 1mol%PEG-c-DOMG, and structure is very equal Matter.Engineer's scale represents 100nm.
Figure 21 by relatively relative FVII protein levels (%) as the DLin-KC2-DMA contents 40mol% in LNP~ The body for the microfluid that LNP in the function illustration factor Ⅴ II mouse models of the siRNA dosage (mg/kg) of 60mol% changes is produced Interior silencing activity.LNP preparations containing 1mol%PEG-c-DOMG and 60mol%DLin-KC2-DMA are provided to be similar to and reported before The FVII silences of the alternative method of the use in road.In 40mol%~60mol% scope, the LNP containing DLin-KC2-DMA Gene silencing progressively improves.Systematicness of the LNP-siRNA to mouse is carried out by tail vein injection (n=3 is per dosage level) Injection.Enter promoting circulation of blood collection after 24 hours after injection and factor Ⅴ II levels are determined by colorimetric estimation.LNP DSPC:Courage Sterol ratio is maintained at 0.2wt/wt and contains 1mol%PEG-c-DOMG.LNP siRNA:Lipid ratio is 0.06wt/wt.
The Cryo electron microscopy inspection for the lipid nanoparticle that Figure 22 A~22C illustrations are prepared by Microfluidics method.It is logical Cross empty lipid nanoparticle (40%DLinKC2-DMA, 11.5%DSPC, 47.5% cholesterine, 1% of Microfluidics preparation PEG-c-DMA) display is indicated inside the electron dense of entity core texture (Figure 22 A).The sample constituted with POPC is shown and water Property cored vesicle association deficient close inside (Figure 22 B).With containing for the triolein hydrophobic core surrounded by POPC individual layer The system of POPC/ trioleins is shown inside the electron dense similar to sample A (Figure 22 C).
Figure 23 illustrations are prepared with DLinKC2-DMA/PEG- lipid systems (90/10, mol/mol) using Microfluidic Mixing Arrowhead LNP is by comparing average grain diameter (nm) as the function of LNP ethanol/water velocity ratio, and LNP has (N/ in siRNA P=1) and siRNA be not present (no siRNA) under produce.Preparation is used and 25mM acetate buffers, the 10mM fat of pH4 mixing Matter-ethanol is mutually carried out.Particle diameter determines and reported the average diameter of number-weighting by dynamic light scattering.
Figure 24 A~24C illustrations are using Microfluidic Mixing in 50%DLinKC2-DMA, 45% cholesterine and 5%PEG-c- The siRNA's wrapped up in DMA31P NMR.Ignore DSPC to avoid the conflict phosphorus signal occurred from phosphatide.Complete LNP or addition It is undetectable from siRNA's after 150mM ammonium acetates31P signal (Figure 24 A) (Figure 24 B).It can only be dissolved in addition 1%SDS Signal (Figure 24 C) is detected after particle.
Figure 25 is the running gel for the result that the protection of illustration RNase is determined.SiRNA uses microfluidic methods (MF) or PFV Method is wrapped up, or keeps not wrapping up.Triton X-100 are added, to be completely dissolved and crack lipid particle.It is natural 20% Gel electrophoresis is carried out on polyacrylamide gel, and siRNA is visualized by using CYBR-SAFE dyeing.
Figure 26 illustrations mix the knot determined as the lipid mixing fusion of the function stand of time (second) using percentage lipid Really.In order to evaluate the amount of the cation lipid of exposure present in LNP outermost layer, 3 LNP systems are prepared：In siRNA missings Under (no siRNA), with N/P=4, and N/P=1.Lipid assay is carried out in pH5.5, to ensure the almost complete of cation lipid Ionization, and react by the way that LNP is injected into containing height anion DOPS/NBD-PE/Rh-PE (98:1:1 mol ratio) vesica Cuvette is originated.
Figure 27 is showing by entity core LNP siRNA systems formed by the method according to the invention Microfluidic Mixing It is intended to.
Figure 28 A and 28B difference illustration average grain diameter (nm) and zeta potential (mV), are used as what is prepared using microfluid mixer The function of continuous lipid nanoparticle composition.
Figure 29 is the schematic diagram of the representative apparatus and method of the invention of the continuous assembling for lipid nanoparticle.
Figure 30 is the schematic diagram of the representative apparatus and method of the present invention.
Figure 31 is the schematic diagram of the representative apparatus and method of the present invention.
Figure 32 is the schematic diagram of the representative apparatus and method of the present invention.
Figure 33 is the schematic diagram of the representative apparatus and method of the present invention.
【Detailed description of the invention】
The present invention provides the lipid particle containing therapeutic agent, the method and apparatus for manufacturing the lipid particle containing therapeutic agent, And the method that therapeutic agent is delivered using lipid particle.
【Lipid particle】
On the one hand, the present invention provides the lipid particle containing therapeutic agent.Lipid particle includes one or more of cationic lipids Matter, one or more of 2nd lipids, and one or more of nucleic acid.
Cation lipid lipid particles include cation lipid.As used herein, term " cation lipid " censure sun from Son or as pH below the pK for being reduced to the ionizable group of lipid turn into cation (protonation), but in higher pH It is worth progressively more neutral lipid.In below pK pH value, then lipid can combine electronegative nucleic acid (for example, oligonucleotides). As used herein, term " cation lipid " includes the amphion lipid of presumption positive charge after pH reductions.
Term " cation lipid " is censured in selective pH, and such as physiology pH carries many lipid matters of net positive charge It is any.The lipid includes, but is not limited to, N, oil base-N, the N- dimethyl ammonium chlorides (DODAC) of N- bis-；N- (the oil of 2,3- bis- Acyloxy) propyl group)-N, N, N- trimethylammonium chlorides (DOTMA)；N, N- distearyl-N, N- Dimethyl Ammonium bromide (DDAB)；N- (2,3- dioleoyls epoxide) propyl group)-N, N, N- trimethylammonium chlorides (DOTAP)；3- (N- (N ', N '-dimethyl Aminoethane)-carbamoyl) cholesterine (DC-Chol) and N- (the myristoyl epoxide propyl- 3- yls of 1,2- bis-)-N, N- diformazans Base-N- hydroxy ethylammonium bromides (DMRIE).In addition, many business prepared products of cation lipid can be used in the present invention.These Including, for example,(commercially available cationic-liposome includes DOTMA and 1,2- dioleoyls-sn-3- Phosphoethanolamine (DOPE), from GIBCO/BRL, Grand Island, NY)；Cationic-liposome(commercially available cationic lipid Plastid includes N- (1- (2,3- dioleoyls epoxide) propyl group)-N- (2- (spermine carboxamido) ethyl)-N, N- Dimethyl Ammonium trifluoros Acetate (DOSPA) and (DOPE), from GIBCO/BRL)；And(commercially available cationic lipid Matter includes two-octadecylamino glycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, WI). Following lipid is cation and has positive charge in below physiology pH：DODAP, DODMA, DMDMA, 1, the sub- oil base oxygen of 2- bis- Base-N, N- dimethylaminopropanecompounds (DLinDMA), 1,2- bis- flax base epoxide-N, N- dimethylaminopropanecompounds (DLenDMA).
In one embodiment, cation lipid is amino lipids.Suitable amino lipids bag useful in the present invention Those for being described in WO 2009/096558 are included, it is incorporated herein by reference in their entirety.Represent amino lipids sub- including 1,2- bis- Oil base epoxide -3- (dimethylamino) acetoxy-propane (DLin-DAC), 1,2- bis- sub- oil base epoxide -3- morpholino propane (DLin-MA), the Asia oleoyl -3- dimethylaminopropanecompounds of 1,2- bis- (DLinDAP), 1, the 2- bis- thio -3- dimethylaminos of sub- oil base Base propane (DLin-S-DMA), the sub- oil base epoxide -3- dimethylaminopropanecompounds (DLin-2-DMAP) of l- Asias oleoyl -2-, 1,2- bis- Sub- oil base epoxide -3- trimethylammoniopropans chloride salt (DLin-TMACl), 1,2- bis- Asia oleoyl -3- trimethylaminos third Alkane chloride salt (DLin-TAPCl), 1,2- bis- sub- oil base epoxide -3- (N methyl piperazine is simultaneously) propane (DLin-MPZ), 3- (the sub- oil base amino of N, N- bis-) -1,2-PD (DLinAP), 3- (the oil base amino of N, N- bis-) -1,2-PD (DOAP), 1, The sub- ethoxy propanes (DLin-EG-DMA) of oil base oxo -3~(2N, N- dimethylamino) of 2- bis- and the sub- oil base -4- two of 2,2- bis- Methylaminomethyl-[l, 3]-dioxolane (DLin-K-DMA).
Suitable amino lipids include having those of the formula：
Wherein R1And R2It is identical or different, and be independently：Optionally substituted C10~C24Alkyl, optionally substituted C10 ~C24Alkenyl, optionally substituted C10~C24Alkynyl, or optionally substituted C10~C24Acyl group；
R3And R4It is identical or different, and be independently：Optionally substituted C1~C6Alkyl, optionally substituted C2~C6Alkene Base, or optionally substituted C2~C6Alkynyl or R3And R4It is engageable and form optionally substituted 4~6 carbon atoms and 1 or 2 The individual heteroatomic heterocycle selected from nitrogen and oxygen；
R5Missing is present, and is hydrogen or C when it is present1~C6Alkyl；
M, n and p are identical or different, and are independently：0 or 1, on condition that, m, n is 0 when different with p；
Q is 0,1,2,3 or 4；And
Y and Z are identical or different, and are independently：O, S or NH.
In one embodiment, R1And R2It is respectively sub- oil base, and amino lipids are two sub- oil base amino lipids.Implement one In mode, amino lipids are two sub- oil base amino lipids.
Representational two useful sub- oil base amino lipids have following formula：
Wherein n is 0,1,2,3 or 4.
In one embodiment, cation lipid is DLin-K-DMA.In one embodiment, cation lipid is (2) more than DLin-K-DMA, wherein n are to DLin-KC2-DMA.
Other suitable cation lipids include, and the cation lipid of net positive charge are carried in about physiology pH, except upper State those it is special outside, N, oil base-N, the N- dimethyl ammonium chlorides (DODAC) of N- bis-；N- (the oil base epoxides of 2,3- bis-) propyl group-N, N-N- triethyl ammoniums chloride (DOTMA)；N, N- distearyl-N, N- Dimethyl Ammonium bromide (DDAB)；N- (2,3- dioleoyls Epoxide) propyl group)-N, N, N- trimethylammonium chlorides (DOTAP)；Oil base epoxide -3- trimethylammoniopropan the chlorides of 1,2- bis- Salt (DOTAPCl)；3 β-(N- (N', N'- dimethylamino ethane) carbamoyl) cholesterine (DC-Chol), N- (1- (2, 3- dioleoyls epoxide) propyl group)-N-2- (spermine carboxamido) ethyl)-N, N- Dimethyl Ammoniums trifluoroacetate (DOSPA), two- Octadecylamino glycyl carboxyspermine (DOGS), 1,2- dioleoyl -3- dimethylammonium propanes (DODAP), N, N- dimethyl - 2,3- dioleoyls epoxide) propylamine (DODMA) and N- (the myristoyl epoxide propyl- 3- yls of 1,2- bis-)-N, N- dimethyl-N-hydroxies Ammonium bromide (DMRIE).In addition, many business prepared products of cation lipid can be used, such as, for example, LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL), and cationic-liposome (DOSPA and DOPE being included, available from GIBCO/BRL).
Cation lipid is present in lipid particle with the amount of about 30~about 95 molar percentages.In one embodiment, Cation lipid is present in lipid particle with the amount of about 30~about 70 molar percentages.In one embodiment, cationic lipid Matter is present in lipid particle with the amount of about 40~about 60 molar percentages.
In one embodiment, lipid particle include only one or more of cation lipids and one or more of nucleic acid (" by It is constituted ").The preparation for the lipid particle of the invention being made up of cation lipid and nucleic acid and it is characterized in described in embodiment 5.
In certain embodiments, lipid particle includes one or more of 2nd lipids to 2nd lipid.The 2nd suitable lipid Particle during stabilizing their formation is formed.
Term " lipid " denotion is the ester of aliphatic acid, and is characterised by insoluble in water but solvable in many organic solvents One group of organic compound.Lipid is generally divided at least 3 classes：(1) " simple lipid ", it includes fat and oil and wax；(2)" Compound lipids ", it includes phosphatide and glycolipid；And (3) " derivative lipid " such as steroid.
Suitable stabilisation lipid includes neutral lipid and anion lipid.
Neutral lipid terms " neutral lipid " are censured with uncharged or neutral zwitterionic form is present in physiology pH Any one of many lipid matters.Representing neutral lipid includes diacyl phosphatidyl choline, diacyl phosphatidyl ethanol Amine, ceramide, sphingomyelins, dihydro sphingomyelins, cephalin and cerebroside.
Illustrating lipid includes, for example, DSPC (DSPC), DOPC (DOPC), two Palmitoylphosphatidyl choline (DPPC), DOPG (DOPG), DPPG (DPPG), two oil Acyl-phosphatidyl-ethanolamine (DOPE), POPC (POPC), palmitoyl-oleyl-phosphatidyl-ethanolamine And dioleoyl-phosphatidyl-ethanolamine 4- (N- maleimidomehyls)-hexamethylene -1- carboxylic acids { salt | ester } (DOPE- (POPE) Mal), DPPE (DPPE), two myristoyl phosphoethanolamines (DMPE), distearyl-phosphatidyl second Hydramine (DSPE), 16-O- monomethyls PE, the 16-O- dimethyl trans PE of PE, 18-1-, 1- stearoyls -2- oleoyl-phosphatidyl ethanol Amine (SOPE) and the anti-Oleoyl-sn-Glycero -3- phosphoethanolamines (transDOPE) of 1,2- bis-.
In one embodiment, neutral lipid is DSPC (DSPC).
Anion lipid terms " anion lipid " are censured in the electronegative any lipids of physiology pH.These lipid bags Include phosphatidyl glycerol, cuorin, diacyl phosphatidyl serine, diacyl phosphatidic acids, N- dodecanoyls phosphatidyl ethanol-amine, N- succinyl phosphatidyl-ethanolamines, N- glutaryl phosphatidyl-ethanolamines, lysyl phosphatidyl glycerol, palmitoyloleoyl phosphatidyl is sweet Oily (POPG), and it is joined to other anion modified groups of neutral lipid.
For example, other suitable lipids include glycolipid (monosialoganglioside GM1).Other the 2nd suitable lipids Including sterol, such as cholesterine.
In certain embodiments, the 2nd lipid is polyethylene glycol-lipid to polyethylene glycol-lipid.Suitable polyethylene glycol- The phosphatidyl-ethanolamine that lipid is modified including PEG-, the phosphatidic acid of PEG- modifications, the ceramide of PEG- modifications is (for example, PEG- CerC14 or PEG-CerC20), the dialkylamine of PEG- modifications, the diacylglycerol of PEG- modifications, the dialkyl group of PEG- modifications Glycerine.Representing polyethylene glycol-lipid includes PEG-c-DOMG, PEG-c-DMA and PEG-s-DMG.In one embodiment, poly- second Glycol-lipid is N- [(methoxyl group PEGs2000) carbamoyl] -1,2- myristyl epoxide propyl group -3- amine (PEG-c-DMA).In one embodiment, polyethylene glycol-lipid is PEG-c-DOMG).
In certain embodiments, the 2nd lipid is present in lipid particle with the amount of about 1~about 10 molar percentage. In one embodiment, the 2nd lipid is present in lipid particle with the amount of about 1~about 5 molar percentage.In one embodiment, 2nd lipid is present in lipid particle with about 1 molar percentage.
The lipid particle of the nucleic acid present invention is useful for the systematicness or local delivery of nucleic acid.It is as described herein, by nucleic acid Lipid particle is incorporated to during its formation.
As used herein, term " nucleic acid " means to include any oligonucleotides or polynucleotides.Containing up to 50 nucleotides Fragment be commonly known as oligonucleotides, and more long segment is referred to as polynucleotides.In certain embodiments, widow of the invention Nucleotides is that 20~50 nucleotides are long.In the scene of the present invention, term " polynucleotides " and " oligonucleotides " are censured by day The alkali so existed, the polymer or oligomer of the nucleotides of (main chain) connection composition or nucleoside monomers between sugar and sugar.Term " multinuclear Thuja acid " and " oligonucleotides " also includes the naturally occurring monomer of the non-comprising similarly function, or part thereof polymer Or oligomer.The modification or substituted oligonucleotides is usually preferred compared to native form, because property is such as, for example, in core Enhanced cellular uptake and increased stability in the presence of sour enzyme.Oligonucleotides is categorized as deoxyribose oligonucleotide or ribose is few Nucleotides.By being referred to as the 5- carbon sugar of deoxyribose, sugared 5 herein ' and 3 ' carbon is covalently joined to phosphoric acid to deoxyribose oligonucleotide And alternating is formed, the polymer composition of non-branch.Ribooligonucleotide is by similar repetitive structure group that wherein 5- carbon sugar is ribose Into.The nucleic acid being present in the lipid particle of the present invention includes any type of nucleic acid known.Nucleic acid used herein can be Single stranded DNA or RNA, or double-stranded DNA or RNA, or DNA RNA hybrid.The example of double-stranded DNA includes structural gene, including control With the gene of terminator, and itself-dubbing system such as virus or DNA.The example of double-stranded RNA includes siRNA and other RNA Interfere reagent.Single-chain nucleic acid includes ASON, ribozyme, microRNA and triplet-formation oligonucleotides.
In one embodiment, polynucleotide is ASON.In certain embodiments, nucleic acid is antisensenucleic acids, Ribozyme, tRNA, snRNA, siRNA, shRNA, ncRNA, miRNA, pre- condensing DNA, or it is fit.
Term " nucleic acid " also censures ribonucleotide, deoxynucleotide, the ribonucleotide of modification, the deoxyribose of modification Nucleotides, phosphoric acid-sugar-backbone oligonucleotides of modification, other nucleotides, nucleotide analog and its combine, and can be single-stranded, Double-strand or the part containing double-strand and single stranded sequence, if suitably.
Term " nucleotides ", as used herein, generically including following term defined below：Nucleosides soda acid, nucleosides, core Thuja acid analog, and universal nucleotide.
Term " nucleotide base ", as used herein, censures Parent Aromatic ring that is substitution or being unsubstituted.In some realities Apply in mode, aromatic ring contains at least one nitrogen-atoms.In some embodiments, nucleotide base can be with appropriate complementary nucleotide Soda acid base formation Watson-Crick and/or Hoogsteen hydrogen bonds.Illustrating nucleotide base and its analog includes, but does not limit It is formed on, purine such as 2- diaminopurines, 2,6- diaminopurines, adenine (A), ethenylidene adenine, N6-2- iso-amylene Base adenine (6iA), N6-2- isopentene group -2- isopentenyl gland purines (2ms6iA), N6- methyl adenines, guanine (G), Isoguanine, N2- dimethylguanines (dmG), 7- methyl guanines (7mG), 2- thiopyrimidines, 6-thioguanine (6sG) Hypoxanthine and O6-MG；7- denitrogenations-purine such as 7- denitrogenations adenine (7- denitrogenations-A) and 7- deazaguanines (7- denitrogenations-G)；Pyrimidine such as cytimidine (C), 5- propynylcytosines, iso-cytosine, thymidine (T), the thio thymus gland of 4- is phonetic Pyridine (4sT), 5,6- dihydrothymines, O4- METHYLTHYMINEs, uracil (U), 4- paper substrates (4sU) and 5,6- bis- Hydrogen uracil (dihydrouracil；D)；Indoles such as nitroindoline and 4- methyl indols；Pyrroles's such as nitro-pyrrole；Pigment gill fungus Element；Base (Y)；In some embodiments, nucleotide base is universal nucleotide base.Extra illustration nucleotide base can Fasman, 1989, Practical Handbook of Biochemistry and Molecular Biology are seen, Pp.385-394, CRC Press, Boca Raton, Fla., and the bibliography that this article is quoted.The further example of universal base Such as Loakes, N.A.R.2001, vol 29 can be found in:2437-2447 and Seela N.A.R.2000, vol 28:3224- 3232。
Term " nucleosides ", as used herein, censures the change of the nucleotide base with the C-1' carbon for being covalently attached to pentose Compound.In some embodiments, connection is through heteroaromatic rings nitrogen.Typical pentose includes, but is not limited to, one or more carbon Those pentoses that atom is respectively independently replaced with one or more identicals or difference-R ,-OR ,-NRR or halogen group, wherein Each R is independently hydrogen, (C1~C6) alkyl or (C5~C14) aryl.Pentose saturable or unsaturation.Illustrate pentose similar with its Thing includes, but is not limited to, ribose, 2'- deoxyriboses, 2'- (C1~C6) alkoxy ribose, 2'- (C5~C14) aryloxy group core Sugar, 2', 3 '-dideoxy ribose, 2', 3 '-two dehydrogenation ribose, 2'- -3 '-haloriboses of deoxidation, 2'- deoxidation -3 '-fluorine ribose, 2'- deoxidation -3 '-chloro ribose, 2'- deoxidation -3 '-amino ribose, 2'- deoxidations -3 '-(C1~C6) alkylribose, 2'- deoxidations - 3 '-(C1~C6) alkoxy ribose and 2'- deoxidations -3 '-(C5~C14) aryloxy group ribose.Also see, for example, 2'-O- methyl, The different head nucleotides of 4'-. α .-, the different head nucleotides of 1'-. α .- (Asseline (1991) Nucl.Acids Res.19:4067-74), 2'-4'- and 3 ' -4'- connections and other " locks " or " LNA ", bicyclic sugar-modified (WO 98/22489；WO 98/39352；WO 99/14226)." LNA " or " lock nucleic acid " be conformation lock cause ribose ring by 2'- oxygen and 3 '-or 4'- carbon between methylene The DNA analogs of base connection constraints.Being connected the conformation limitation forced usually increases binding affinity and the increasing to complementary series Plus the heat endurance of the doublet.
Sugar is included in 2'- or 3 ' modification of-position, such as methoxyl group, ethyoxyl, allyloxy, isopropoxy, fourth oxygen Base, isobutoxy, methoxy ethyl, alkoxy, phenoxy group, azido, amino, alkylamino, fluorine, chlorine and bromine.Nucleosides and nucleosides Acid include natural D configurational isomers (D- types), and L-configuration isomers (L-type) (Beigelman, U.S.Pat.No.6, 251,666；Chu,U.S.Pat.No.5,753,789；Shudo,EP0540742；Garbesi(1993)Nucl.Acids Res.21:4159-65；Fujimori(1990)J.Amer.Chem.Soc.112:7435；Urata,(1993)Nucleic Acids Symposium Ser.No.29:69-70).When core base is purine, for example, A or G, ribose sugar attaches to core alkali The N9- positions of base.When core base is pyrimidine, for example, C, T or U, pentose attach to the N1- positions (Kornberg of core base and Baker,(1992)DNA Replication,2.sup.nd Ed.,Freeman,San Francisco,Calif.)。
One or more pentose carbon phosphoric acid esters substitution of nucleosides.In some embodiments, phosphates attach to penta 3 '-or 5 '-carbon of sugar.In some embodiments, nucleosides is that nucleotide base is purine, 7- deazapurines, pyrimidine, general purpose core Those of thuja acid base, specific nucleotide base, or its analog.
Term " nucleotide analog ", as used herein, censures pentose and/or nucleotide base and/or one of nucleosides Or more phosphate can with its corresponding analogs replace embodiment.In some embodiments, illustrating pentose sugar analog is Those described above.In some embodiments, nucleotide analog has above-mentioned nucleobase analog.In some embodiments In, illustrating phosphate analogs includes, but is not limited to, phosphonate ester, methyl phosphonate, phosphoramidate, phosphotriester, Thiophosphate, phosphorodithioate, phosphoroselenoate, two phosphoroselenoates, anilino- thiophosphate, anilino- phosphoric acid Ester, phosphoramidate, borane phosphonate with may include to associate to ion.Other nucleic acid analogs and base include for example being embedded in Nucleic acid (INA is such as described in Christensen and Pedersen, 2002) and AEGIS bases (Eragen, United States Patent (USP) No.5, 432,272).The extra description of various nucleic acid analogs is also seen in such as (Beaucage et al., Tetrahedron 49(10):Bibliography in 1925 (1993) and this article；Letsinger,J.Org.Chem.35:3800(1970)； Sprinzl et al.,Eur.J.Biochem.81:579(1977)；Letsinger et al.,Nucl.Acids Res.14: 3487(1986)；Sawai et al,Chem.Lett.805(1984),Letsinger et al., J.Am.Chem.Soc.110:4470(1988)；and Pauwels et al.,Chemica Scripta 26:141 91986)), thiophosphate (Mag et al., Nucleic Acids Res.19:1437(1991)；and U.S.Pat.No.5,644,048).Other nucleic acid analogs comprising phosphorodithioate (Briu et al., J.Am.Chem.Soc.111:2321 (1989)), O- methyl phosphoramidite keys are (see Eckstein, Oligonucleotides and Analogues:A Practical Approach, Oxford University Press), with that of positive backbones (Denpcy et al., Proc.Natl.Acad.Sci.USA 92 a bit:6097(1995))；Non- ion main chain (U.S.Pat.Nos.5,386,023,5,386,023,5,637,684,5,602,240,5,216,141,and 4,469, 863.Kiedrowshi et al.,Angew.Chem.Intl.Ed.English 30:423(1991)；Letsinger et al.,J.Am.Chem.Soc.110:4470(1988)；Letsinger et al.,Nucleoside&Nucleotide 13: 1597(194):Chapters 2 and 3,ASC Symposium Series 580,"Carbohydrate Modifications in Antisense Research",Ed.Y.S.Sanghui and P.Dan Cook；Mesmaeker et al.,Bioorganic&Medicinal Chem.Lett.4:395(1994)；Jeffs et al.,J.Biomolecular NMR 34:17(1994)；Tetrahedron Lett.37:743 (1996)) and non-ribose backbone, including to be described in the U.S. special Sharp No.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, " Carbohydrate Modifications in Antisense Research",Ed.Y.S.Sanghui and P.Dan Those of Cook.Containing one or more of carbocyclic rings sugar nucleic acid be also included within the definition of nucleic acid (see Jenkins et al., Chem.Soc.Rev.(1995)pp169-176).Several nucleic acid analogs are also to be described in Rawls, C＆E News 1997 6 Page 35 of the moon 2.
Term " universal nucleotide base " or " universal base ", as used herein, denotion, which can contain or can be free of, nitrogen-atoms Aromatic ring component.In some embodiments, universal base can covalently be attached to the C-1' carbon of pentose and manufactures general purpose core Thuja acid.In some embodiments, universal nucleotide base not especially with another nucleotide base hydrogen bonding.In some implementations In mode, universal nucleotide base and nucleotide base hydrogen bonding reach and including complete nucleotide base especially target multinuclear Thuja acid.In some embodiments, nucleotide base can pass through hydrophobic heap with the adjacent nucleotide base in identical nucleic acid chains Folded interaction.Universal nucleotide includes, but is not limited to, deoxidation -7- azaindoles triphosphoric acid (d7AITP), deoxidation isoquinoline promise Ketone triphosphoric acid (dICSTP), deoxidation propinyl isoquinolone triphosphoric acid (dPICSTP), deoxidation methyl -7- azaindole triphosphoric acids (dM7AITP), deoxyImPy triphosphoric acids (dImPyTP), deoxyPP triphosphoric acids (dPPTP), or deoxidation propinyl -7- azepines Yin Diindyl triphosphoric acid (dP7AITP).The further example of the universal base can especially see disclosed U. S. application No.10/290672, and United States Patent (USP) No.6,433,134.
As used herein, term " polynucleotides " and " oligonucleotides " used interchangeably, and refer to the single-stranded of nucleotide monomer And double-chain polymer, including 2'- deoxyribonucleotides (DNA) and ribonucleotide (RNA), it passes through internucleotide phosphate two Ester bond is connected, for example, 3 ' -5 ' and 2'-5', inverted connection, for example, 3 ' -3 ' and 5 ' -5', the structure of branch, or between nucleotides Analog is connected.Polynucleotides are associated with to ion, such as H+, NH4+, trialkyl ammonium, Mg2+, Na+, etc..Polynucleotides can be complete It is made up of, is made up of completely ribonucleotide deoxyribonucleotide, or its chimeric mixtures.Nucleosides between polynucleotides can be included Acid, core base and/or sugar analogue.Polynucleotides are typically dimensionally from few monomeric unit, for example, when they are in ability It is 3~40 when being more generally often referred to as oligonucleotides in domain, to thousands of monomeric nucleotide units.Unless indicated otherwise, no matter When polynucleotide sequence is represented, nucleotides is represented from left to right with 5 ' to 3 ' orders, and " A " represents desoxyadenossine, " C " table Show dideoxycytosine, " G " represents that deoxyguanosine, and " T " represent thymidine, unless otherwise defined.
As used herein, " core base " refers to those naturally occurring and naturally occurring utilization nucleic acids of those non- Or the generally known heterocycle of people for the polymer that can be specifically bound with nucleotide sequence is thus produced using peptide nucleic acid technology Part.The non-limiting example of suitable core base includes：Adenine, cytimidine, guanine, thymidine, uracil, 5- propine Base-uracil, the thio -5- propinyl-uracils of 2-, 5-methylcytosine, false iso-cytosine, 2- paper substrates and 2- are thio Thymidine, 2- diaminopurines, N9- (2- amino -6- chloro-purines), N9- (2,6- diaminopurine), hypoxanthine, N9- (7- denitrogenations-guanine), N9- (7- denitrogenations -8- azepines-guanine) and N8- (7- denitrogenations -8- azepines-adenine).Suitable core Other non-limiting examples of base are included in Buchardt et al. the Fig. 2 (A) and 2 (B) of (WO92/20702 or WO92/20703) Those core bases of illustration.
As used herein, " core base sequence " refers to the aggregation (such as 2 or more of any section, or 2 or more section The aggregation core base sequence of oligomer block), the aggregation of the polymer comprising the subunit containing core base.Suitable polymer or The non-limiting example of polymer segments includes oligodeoxynucleotide (such as DNA), oligoribonucleotide (such as RNA), peptide nucleic acid (PNA), PNA chimeras, PNA combination oligomer, nucleic acid analog and/or nucleic acid mimics.
As used herein, " many core base chains " refers to the complete single polymer chain for including core base subunit.For example, double-strand core The mononucleotide chain of acid is many core base chains.
As used herein, " nucleic acid " is the polymer containing core base sequence, or polymer segments, and it has by nucleotides, Or the main chain of its analog formation.
It is preferred that nucleic acid be DNA and RNA.
As used herein, nucleic acid can also censure " peptide nucleic acid " or " PNA " and refer to comprising 2 or more PNA subunits (residual Thing), but be not any oligomer or polymer segments (such as block oligomer) of nucleic acid subunit (or its analog), including but not It is limited in following documents to be referred to as peptide nucleic acid or any oligomer or polymer segments as peptide nucleic acid requirement：United States Patent (USP) No.5,539,082,5,527,675,5,623,049,5,714,331,5,718,262,5,736,336,5,773,571,5, 766,855,5,786,461,5,837,459,5,891,625,5,972,610,5,986,053 and 6,107,470；By drawing With being incorporated herein.Term " peptide nucleic acid " or " PNA " should be also applied to be described in the bag of those nucleic acid mimics of following publications Any oligomer or polymer segments containing 2 or more subunits：Lagriffoul et al.,Bioorganic&Medicinal Chemistry Letters,4:1081-1082(1994)；Petersen et al.,Bioorganic&Medicinal Chemistry Letters,6:793-796(1996)；Diderichsen et al.,Tett.Lett.37:475-478 (1996)；Fujii et al.,Bioorg.Med.Chem.Lett.7:637-627(1997)；Jordan et al., Bioorg.Med.Chem.Lett.7:687-690(1997)；Krotz et al.,Tett.Lett.36:6941-6944 (1995)；Lagriffoul et al.,Bioorg.Med.Chem.Lett.4:1081-1082(1994)；Diederichsen, U.,Bioorganic&Medicinal Chemistry Letters,7:1743-1746(1997)；Lowe et al., J.Chem.Soc.Perkin Trans.1,(1997)1:539-546；Lowe et J.Chem.Soc.Perkin Trans.11: 547-554(1997)；Lowe et al.,J.Chem.Soc.Perkin Trans.11:555-560(1997)；Howarth et al.,J.Org.Chem.62:5441-5450(1997)；Altmann,K-H et al.,Bioorganic&Medicinal Chemistry Letters,7:1119-1122(1997)；Diederichsen,U.,Bioorganic& Med.Chem.Lett.,8:165-168(1998)；Diederichsen et al.,Angew.Chem.Int.Ed.,37:302- 305(1998)；Cantin et al.,Tett.Lett.,38:4211-4214(1997)；Ciapetti et al., Tetrahedron,53:1167-1176(1997)；Lagriffoule et al.,Chem.Eur.J.,3:912-919 (1997)；Kumar et al.,Organic Letters 3(9):1269-1272(2001)；And such as it is disclosed in WO96/ 04000 Shah et al. Peptide-Based Nucleic Acid Mimics (PENAMS).
【Lipid particle feature】
The lipid particle of the form present invention is its form different from other materials being similarly formed, and is characterized as tool There is substantially entity core.Lipid particle with substantially entity core is the aqueous area internally without extension and had The mainly particle of the inside of lipid.In one embodiment, the area of extension is that volume is more than the continuous aqueous of half particle volume Area.In the 2nd embodiment, the aqueous area of extension is more than 25% particle volume.The degree in internal aqueous area can pass through electronics Microscope inspection determines and is rendered as the area of low electron density.Moreover, because the inside of entity core nanoparticles is mainly lipid, The lipid of water content (" volume of capture ")/constituent particle of particle is less than the individual layer lipid bilayer vesica with identical radius It is expected.In one embodiment, the volume of capture is less than expected from the 50% individual layer bilayer vesicle with identical radius. In the 2nd embodiment, the volume of capture is less than expected from the individual layer bilayer vesicle of 25% identical size.In the 3rd embodiment party In formula, the volume of capture is less than the cumulative volume of 20% particle.In one embodiment, volume/lipid of capture is less than 2 μ l/ μ Mol lipids.In another embodiment, the volume of capture is less than 1 μ l/ μm ol lipids.In addition, when with the increase of vesica radius When volume/lipid of the capture of lipid bilayer vesica substantially increases, as the radius of entity core nanoparticles increases and catch Volume/the lipid obtained does not increase substantially.In one embodiment, as average-size is from 20nm diameter to the straight of 100nm The increase in footpath and the volume/lipid increase that captures is less than 50%.In the 2nd embodiment, as average-size is from the straight of 20nm To the increase of 100nm diameter, the volume/lipid increase that captures is less than 25% in footpath.The volume of capture can use and be described in document Various technologies measurement.Because entity core system contains lipid, the given radius of every mole of lipid generation within particle Total number of particles be less than bilayer vesicle system expected from.The population that every mole of lipid is produced can be surveyed by especially fluorescent technique Amount.
The lipid particle of the present invention can also be characterized by Electron microscopy.The particle of the present invention, which has substantially to have, to be led to Cross the entity core for the electron-dense cores that Electron microscopy is seen.Electron dense is defined as the inside of entity core particle The electron density (as seen in 2-D freezings EM as in) in the area in the area of 50% projection-average not less than x% (x=20%, 40%, 60%) in the maximum electron density in particle periphery.Electron density calculates the image intensity for purpose area with not containing nanometer The absolute value of the difference of background intensity in the area of particle.
The lipid particle of the particle diameter present invention has about 15~about 300nm diameter (average grain diameter).In some embodiments In, lipid particle has about 300nm or smaller, 250nm or smaller, 200nm or smaller, 150nm or smaller, 100nm or smaller, Or 50nm or less diameter.In one embodiment, lipid particle has about 15~about 100nm diameter.These particles lead to Often present and compare the increased circulation lifetime in vivo of big particle.In one embodiment, lipid particle has about 15~about 50nm's Diameter.These particles can advantageously escape out vascular system.In one embodiment, lipid particle has about 15~about 20nm's Diameter.Arrowhead of these particles close to the particle containing nucleic acid；For example, (particle may include single polynucleotides siRNA)。
The lipid particle of the present invention substantially homogeneous in their size distribution.In certain embodiments, it is of the invention Lipid particle have about 65~about 25% average grain diameter standard deviation.In one embodiment, lipid particle of the invention With about 60,50,40,35 or 30% average grain diameter standard deviation.
The lipid particle of the parcel efficiency present invention can be distinguished also in parcel efficiency.As described below, liposome of the invention Son by used in forming process almost 100% nucleic acid be wrapped in method in particle and prepare.In one embodiment, It is prepared by the method that lipid particle is wrapped in by about 90~about 95% nucleic acid used in forming process in particle.
【Manufacture the microfluidic methods of lipid particle】
On the one hand, the method that the present invention provides lipid particle of the manufacture containing therapeutic agent.In one embodiment, method bag Include：
For example, (a) the 1st conductance that therapeutic agent (polynucleotide) will be included in the 1st solvent enters microchannel；Wherein microchannel has Have suitable for one or more conductances to be flowed into the 1st area of microchannel and for the 2nd area of the content for mixing one or more streams；
(b) the 2nd conductance for including lipid particle-forming material in the 2nd solvent is entered microchannel to provide in laminar flow article Flowed under part the 1st and the 2nd stream, wherein lipid particle-forming material includes ionizable lipid, and the wherein the 1st and the 2nd is molten Agent is differed；
(c) one or more 1st streams and one or more 2 streams are made to flow into the 2nd of microchannel from the 1st area of microchannel Area；And
(d) one or more 1 flowed under laminar flow condition are blended in the 2nd area of microchannel to flow and one or more The content of 2nd stream flows with the provide the lipid particle for including the therapeutic agent with parcel the 3rd.
The content of 1st and the 2nd stream can be mixed by mixed and disorderly advection.In one embodiment, the one or more 1st is mixed The content of stream and one or more 2 streams includes changing the concentration or phase of one or more 1st streams and one or more 2 streams To mixing velocity.Different from the method known in embodiment of above, method does not include diluting after mixing.
In order to further stabilize the 3rd stream of the lipid particle containing the therapeutic agent with parcel, method can, but be not necessarily to Also include, including flowed with aqueous buffered dilution agent the 3rd.In one embodiment, the stream of dilution the 3rd includes making the 3rd stream and aqueous slow Electuary flows into the 2nd mixed structure.In another embodiment, dialysis bag contains the aqueous of the lipid particle of the therapeutic agent with parcel Buffer is to reduce the amount of the 2nd solvent.
1st stream includes therapeutic agent in the 1st solvent.The 1st suitable solvent include therapeutic agent be it is soluble and with the 2nd solvent Miscible solvent.The 1st suitable solvent includes aqueous buffer.Representing the 1st solvent includes citrate and acetate buffer.
2nd stream includes lipid particle-forming material in the 2nd solvent.The 2nd suitable solvent includes ionizable lipid Be it is soluble and with the solvent of the 1st solvent miscibility.The 2nd suitable solvent includes the dioxane of Isosorbide-5-Nitrae-, tetrahydrofuran, acetone, second Nitrile, dimethyl sulfoxide (DMSO), dimethylformamide, acid and alcohol.Representing the 2nd solvent includes aqueous ethanol 90%.
The method of the present invention is different from other Microfluidic Mixing methods in several ways.But the specific method needs known Equivalent or aqueous solvent and organic solvent (that is, 1 substantially etc. in proportion:1), method of the invention is generally using more than 1:1 Aqueous solvent to the solvent ratio of organic solvent.In certain embodiments, aqueous solvent is about to the solvent ratio of organic solvent 1:2.In certain embodiments, aqueous solvent is about 1 to the solvent ratio of organic solvent:3.In certain embodiments, it is aqueous Solvent is about 1 to the solvent ratio of organic solvent:4.In specific other embodiment, solvent ratio of the aqueous solvent to organic solvent It is about 1:5, about 1:10, about 1:50, about 1:100, or it is bigger.
The lipid particle of the present invention is advantageously formed in the microfluidic methods using relatively rapid mixing and high flow rate.It hurry up Speed mixing provides the lipid particle with above-mentioned favourable property (including size, homogeney, wrap up efficiency).The present invention Method practice in the μ s of mixing velocity scope about 100~about 10ms for using.Representative mixing velocity includes about 1~about 5ms. But hydraulic current focus method with relatively low lipid volume with relative low flow velocity (for example, 5~100 μ L/ minutes) operation, this hair The high lipid volume operation with relative high flow rate and relatively of bright method.In certain embodiments, for merging single mixed zone Method (that is, blender), flow velocity is about 1mL/min.For the side of the invention using blender array (for example, 10 blender) Method, using the flow velocitys of 40mL/ minutes (for 100 blenders, flow velocity 400mL/min).Thus, method of the invention can easily contract Put to provide the amount of lipid particle necessary to require generation demand.The favourable particle diameter and homogeney and parcel for being coupled to realization are imitated Rate, method of the invention overcomes the shortcoming for the microfluidic methods known for producing lipid particle.The manufacture liposome of the present invention One advantage of the method for son is that method is scalable, and this refers to that method does not change and during scaling during scaling There is excellent correspondence.
【Manufacture the microfluidic device of lipid particle】
On the other hand, the present invention provides the device for the lipid particle for producing parcel nucleic acid.Device in one embodiment Including：
(a) the 1st entrance, it is used to receive the 1st solution for including nucleic acid in the 1st solvent；
(b) the 1st inlet microchannel, it circulates with the 1st entrance is connected to provide the 1st stream comprising nucleic acid in the 1st solvent；
(c) the 2nd entrance, it is used to receive the 2nd solution for including lipid particle-forming material in the 2nd solvent；
(d) the 2nd inlet microchannel, it is connected with the circulation of the 2nd entrance and lipid particle-shape is included in the 2nd solvent to provide Into the 2nd stream of material；
(e) the 3rd microchannel, its be used for receive the 1st and the 2nd flow, wherein the 3rd microchannel have be suitable under laminar flow condition will 1st and the 2nd conductance flows into the 1st area of microchannel and the 2nd area of the content flowed suitable for mixing the 1st and the 2nd and includes tool to provide There is the 3rd stream of the lipid particle of the nucleic acid of parcel.
In one embodiment, device also include be used for dilute the 3rd stream facility with provide include it is stabilized have bag The stream of the dilution of the lipid particle for the therapeutic agent wrapped up in.
The device of the present invention is that microfluidic device (that is, is less than 1mm maximum chi including one or more microchannels with it The passage of degree).In one embodiment, microchannel has about 20~about 300 μm of hydrodynamic diameter.As described above, microchannel With 2 areas：For example, for receiving and being flowed under laminar flow condition at least two stream, (one or more 1 flow and one in 1st area Or more the 2nd stream).The content of 1st and the 2nd stream is mixed in the 2nd area of microchannel.In one embodiment, the 2nd of microchannel Area has main flow direction and one or more surfaces, and there is at least one to be defined in groove therein or projection on the surface, recessed Groove or projection have the orientation (for example, herringbone blender staggeredly) with principal direction angulation, are such as described in U. S. application and disclose No.2004/0262223, it is incorporated herein by reference in their entirety.In one embodiment, the 2nd area of microchannel includes basse-taille Structure.In order to reach maximum mixing velocity, it is favourable to avoiding the excess fluid resistance before mixed zone.Thus, this hair A bright embodiment is the logical for fluid to be delivered into single mixing of the non-microfluidic channel with the yardstick more than 1000 μm The device in road.
In other aspects of the present invention, the 1st and the 2nd stream is mixed with other micro-mixers.Suitable micro-mixer includes Drip blender, T- blenders, zig-zag type blender, multilayer blender or other travelling mixers.
The facility that the mixing of 1st and the 2nd stream also can be used for changing the flow velocity of concentration and relative 1st and the 2nd stream is realized.
In another embodiment, producing the device of the lipid particle of parcel nucleic acid includes being used to receive the 1st and the 2nd stream Microchannel, wherein microchannel, which have, to be suitable under laminar flow condition the 1st and the 2nd conductance flowing into the 1st area of microchannel and suitable for mixed The 2nd area for closing the content of the 1st and the 2nd stream flows with the provide the lipid particle for including the therapeutic agent with parcel the 3rd.It is real herein Apply in mode, it is fluent with the microchannel importing microchannel for not being above-mentioned 1st and the 2nd microchannel by the 1st and the 2nd.
In order to reach maximum mixing velocity, it is favourable to avoiding the excess fluid resistance before mixed zone.Thus, originally One embodiment of invention is the non-microfluidic channel with the yardstick more than 1000 μm for fluid to be delivered into single mixing The device of passage.This device for producing the lipid particle of parcel nucleic acid includes：
(a) single entry microchannel, it is used to receive the 1st solution comprising nucleic acid and the bag in the 2nd solvent in the 1st solvent The 2nd solution containing lipid particle-forming material；
(b) it is suitable to the 2nd area of the content that mixing the 1st and the 2nd is flowed to provide the liposome for including the nucleic acid with parcel 3rd stream of son.
In an embodiment, by the 1st and the 2nd stream is by single entry or passes through 1 without micro--yardstick or 2 Passage, for example, the passage of the yardstick (for example, 1500 or 2000 μm or bigger) with more than 1000 μm imports microchannel.These Adjacent or concentric large-sized passage can be used to import inlet microchannel for passage.
Fig. 1 is the schematic diagram of the representative fluid means of the present invention.Fig. 1 is mentioned, device 100 includes being used to receive molten the 1st The area A of the 1st stream comprising therapeutic agent and the area for receiving the stream comprising lipid particle-forming material in the 2nd solvent in agent B.1st and the 2nd conductance is entered to the area C flowed under laminar flow condition, the area D occurred to wherein quick mixing, then to wherein most End-product, the lipid particle containing therapeutic agent goes out the area E of device.
Fig. 2 is the schematic diagram of the representative fluid means of the present invention, and it is the detail of the apparatus and method of illustration in Fig. 1.Say To Fig. 2, device 200 includes being used to receive including the 1st of the therapeutic agent area A for flowing to microchannel, wherein microchannel in the 1st solvent With the 1st area, it is suitable to flowing (A-a) guiding (A-b) and mixing (A-c) of one or more streams；For receiving in the 2nd solvent In the 2nd stream comprising lipid particle-forming material area B, wherein microchannel has the 1st area, and it is suitable to one or more streams Flow (B-a) guiding (B-b) and mixing (B-c)；Area C boot section A and area B under laminar flow condition stream (C-a) and quick mixing (C-b)；And area D, wherein preparation prepare be used for further handle such as dilute, pH regulation or nano-particle synthesis need other Event, or wherein final product, the lipid particle containing therapeutic agent go out device.
Fig. 3 is the schematic diagram of the representative fluid means of the present invention, and it is the detail of the apparatus and method of illustration in Fig. 2.Say To Fig. 3, device 300 includes being used to receive including the 1st of the therapeutic agent area A for flowing to microchannel, wherein microchannel in the 1st solvent With the 1st area, it is suitable to flowing (A-a) guiding (A-b) and mixing (A-c) of one or more streams；For receiving in the 2nd solvent In the 2nd stream comprising lipid particle-forming material area B, wherein microchannel has the 1st area, and it is suitable to one or more streams Flow (B-a) guiding (B-b) and mixing (B-c)；Area C boot section A and area B under laminar flow condition stream (C-a) and quick mixing (C-b)；Area D, it is used for the 3rd stream for receiving the material including other particle-forming materials comprising Arbitrary Digit, and dilution, pH is adjusted Section, or other events that nano-particle synthesis needs；Area E guide area C and area D under laminar flow condition stream (E-a) and quick mixed Close (E-b)；Area F, wherein preparation prepare to be used for further to handle as diluted, other things that pH regulations or nano-particle synthesis need Part, or wherein final product, the lipid particle containing therapeutic agent go out device.
Fig. 4 is another schematic diagram for representing fluid means (400) of the present invention.Fig. 5 is the representative fluid dress of illustration in Fig. 4 The schematic diagram for the representative array put.
Fig. 6 is another schematic diagram for representing fluid means (600) of the present invention.Fig. 6 is mentioned, it is logical that device 600 includes mixing Road 610a, 610b and 610c.Fig. 7 is the schematic diagram of the representative array of the representative fluid means of illustration in Fig. 6.
The formation of nano-particle can be occurred by the reagent volume of participation compound event, and device in seepage on microfluidic device The rear pressure for the limitation resisted before is limited.Compared to drop or stream focus method, herringbone or multilayer blender unit piece flow velocity Reach the increase of 100~1000 multiples., can array polyhybird device element in order to reach production scale flux.In one embodiment, Each reagent is distributed in individual mixer element using low impedance bus passage.If the impedance of bus run is compared to blender member The impedance of part is insignificant, and the individual flow velocity of the entrance of each blender is identical.Due to the parallel behaviour of multiple mixer elements Make, system impedance, which reduces, causes higher stereometry flux.
This has advantage, and the composite character observed using single mixer element can be maintained in blender array.It is real one Apply in mode, the mixing in each blender array element is realized by the way that multiple conductances are entered into microchannel.In this case, flow It can be mixed by spreading.Streamline width can be by controlling the relative velocity by injection canal (for example, by adjusting these passages Yardstick) change (Fig. 5).In another embodiment, mixing passes through mixed and disorderly advection (herringbone blender staggeredly, SHM) Reach.As depicted in Fig. 7, each mixer element of array can be made up of a series of blenders.By adding element to each array Subset, extra feature can be integrated in microfluidic device in line.The feature may include to dilute on chip, dialysis, pH regulations Or streamline staggeredly is needed, share the stream of identical passage or other events of the stream separated each other by porous mass.One In embodiment, 10mM POPC are dissolved in 100% ethanol in the 1st mixer element of each array subset and delayed with phosphate Fliud flushing (PBS), pH7.4 mixing.The LNP formed after mixing is stabilized by the way that mixture is diluted into 2 times with PBS.
Table 1 compares the granularity of the particle formed on single blender and the blender array being made up of 10 individual blenders Distribution.Overall flow rate by single blender can be 4ml/min, be 50 in the mixing ratio of each intersection:50.Stereometry flux can lead to Crossing 10 blenders of operation repetitive increases by 10 times of cumulative volumes measure flow velocitys for obtaining 40ml/min.When array flux complies with production rule When mould is synthesized, LNP yardsticks are maintained.
The granularity point for the particle that table 1. is formed on single blender and the blender array being made up of 10 individual blenders Cloth.
Single blender
It is average<x>(nm) Standard deviation (nm)
Intensity 73 37.8 1.58
Volume 62.8 32.5
Numbering 25.7 13.3
Blender array
It is average<x>(nm) Standard deviation (nm)
Intensity 72.1 35.1 1.08
Volume 62.7 32.4
Numbering 27.0 13.7
Any combinations of the parallel reagent inlet of Arbitrary Digit, continuous mixing chamber, and branch construct can be used for optimization nanoparticle Sub- production process.This has advantage, and different production process can be accurately controlled and multiple steps of nanoparticle formulation process can Integrate.Example includes, but is not limited to, following：(a) import by constitute the 2 of different (Fig. 8) or identical (Fig. 9) reagents Individual or more entrance and allow independent input control (using include input reagent flow velocity independent control, change input reagent Between, and the ratio between other)；(b) 2 or more blenders in order and allow nano-particle reagent or preparation to handle step Rapid continuous addition (Figure 10) (is assembled, system using the bottom for including the control that addition input reagent is used for nano-particle in order The integration of agent process, such as dilutes, other events that pH regulations or nano-particle synthesis need, and other)；Or (c) input, mixing Room and any combinations of branch construct, Figure 11 and Figure 12 illustrations have the parallel reagent input for changing number and branch's microfluidic structures 2 steps and 3 step blenders (using include production process multiple steps integration, it includes the core of nano-particle reagent Mixing on chip, nano-particle have on coring and growth, chip dilute, dialysis, pH regulation or nano-particle synthesis need other Event).
Figure 13 is the schematic diagram of the representative fluid means of the present invention, and it has multilayer blender.Mention Figure 13, device 1300 Including hybrid channel 1310.Figure 14 is the close up view of the multilayer blender of illustration in Figure 14.
As described above, the method for manufacture lipid micro-/ nano particle was once conventional " inversion " method, wherein more big structure is logical Cross that lipid is scattered in water, be by the polycarbonate filter in the aperture with such as 100nm or alternatively, to use afterwards Hold the division of ultrasonically treated multi-layer vesicles (micron size range) and formed.
The one side lacked in the batch list is the ability for the structure and assembling for accurately controlling each lipid mixture composition. This is even more important, if special component is easily degraded, if exposed to their external environment condition, or if particular ligand must Need to be outside the particle of targeting purpose.For example, can be it is important that produce has net positive or negative surface electricity first with therapeutic agent Lotus is to associate the particle of particular treatment medicine.Then, it may be desired to further handle to assemble completely, by using other lipids Matter wraps up the particle, or modifies its surface characteristics.This can be for example including addition lipid, and to produce net neutral particle, or addition must Need to be in the targeted molecular outside the particle of functional purpose.
In one embodiment, the method for manufacture lipid nanoparticle is included by Charge Correlation, and may also provide treatment Property siRNA (siRNA) parcel carry out the continuous assembling and growth of lipid nanoparticle.The method can be used for changing completely Become from net positive to surface charge characteristics net negative and that vice versa.
The lipid nanoparticle for wrapping up siRNA (- ive) is prepared with about 2 (+ive/-ive) charge ratio.Lipid includes 90mol%DLin-KC2-DMA (+ive) and 10mol%PEG-c-DMA.The diameter of obtained particle is 23nm (Figure 28 A) and tool There is about 7mV positive zeta potential (Figure 28 B).Then by anion lipid so that 4 times of excess are through Microfluidic Mixing and close to cationic lipid Matter.This causes particle diameter to increase to 33nm and is changed into -14mV negative zeta potential.Also further and close extra cation lipid (with DOPS of 4 times of excess before) and then and close DOPS and cause the lasting increase of particle diameter, it is and net just and only between negative zeta potential Change.
Result is obtained by being mixed in single microfluid mixer, reclaims, micro-mixer is then injected into again, with addition One lipid composition.But, single microfluidic device may be designed as producing the particle (Figure 29) in a continuous manner.
Following devices minimize fluid impedance and are entering the interaction between the prelipid substance and aqueous fluid of micro-mixer.
Figure 30 is the representative device 3000 of the present invention and the schematic diagram of method.Figure 30 is mentioned, device 3000 includes：Area A, its In in the 1st solvent comprising multiple nucleic acid the 1st stream enter big width passage (>2mm)；With area B, wherein in the 2nd solvent Comprising lipid particle-forming material stream enter big width passage (>2mm).Lead-In Area C will be flowed, wherein in micro-mixer Occurs quick mixing, then most Zhongdao area D, final product.
Figure 31 is the schematic diagram of the representative apparatus and method of the present invention.Figure 31 is mentioned, device 3100 includes：Area A, wherein In 1st solvent comprising multiple nucleic acid the 1st stream enter big width passage (>2mm)；With area B, wherein being included in the 2nd solvent Lipid particle-forming material stream enter big width passage (>2mm).Lead-In Area C will be flowed, wherein occurring in micro-mixer Quickly mix, then most Zhongdao area D, final product.
Figure 32 is the schematic diagram of the representative apparatus and method of the present invention.Figure 32 is mentioned, device 3200 includes：Area A, wherein In 1st solvent comprising multiple nucleic acid the 1st stream enter big width passage (>2mm)；Area B, wherein the 2nd stream is comprising the 1st solvent Play the effect of the sheath fluid of area A stream；Area C, wherein the stream comprising lipid particle-forming material enters big in the 2nd solvent Width passage (>2mm)；And area D, wherein the 2nd stream plays the effect of the sheath fluid of area C stream comprising the 2nd solvent, by conductance Enter area E, mixed wherein generation is quick in micro-mixer, then most Zhongdao area F, final product.Dotted line represents fluid boundary.
Figure 33 is the schematic diagram of the representative apparatus and method of the present invention.Figure 33 is mentioned, device 3300 includes：Area A, wherein The 1st stream comprising multiple nucleic acid and the comprising lipid particle-forming material in the 2nd solvent the 2nd flows to heart inflow in 1st solvent Passage, Lead-In Area B is mixed, then most Zhongdao area C wherein generation is quick in micro-mixer, and wherein we have final product. 2 fluids in area A can isolate by physical barriers or by sheath fluid shown in X-section figure.
【The method that therapeutic agent is delivered using lipid particle】
The lipid particle of the present invention can be used for therapeutic agent is external or delivering in vivo is to cell.In certain embodiments, Therapeutic agent is nucleic acid, and it is used into nucleic acid-lipid particle delivery of the invention to cell.Method and composition can be easily adapted to use In the delivering of any suitable therapeutic agent of the treatment for any disease or illness that can benefit from the treatment.
In certain embodiments, the present invention is provided the method for nucleic acid into cells.It is preferably used in and imports cell Nucleic acid is siRNA, miRNA, immune-stimulating oligonucleotides, plasmid, antisense and ribozyme.These methods can be by making the present invention's The time of Intracellular delivery occurs enough with cells contacting for particle or composition to carry out.
Typical case is applied including the use of well known process to provide siRNA Intracellular delivery, with knockout or silence certain detail Born of the same parents' target.Alternatively application includes the DNA or mRNA sequence of the useful polypeptide of delivering coding treatment.In this way, supply is passed through The gene outcome of defect or shortage and provide treatment be used for genetic disease.The method of the present invention can be external, in vitro or practice in vivo. For example, the composition of the present invention can also be used for nucleic acid using method known to those skilled in the art delivering in vivo to cell.
The delivering for the following describing the siRNA of the lipid particle by the present invention is effective in cryptiogene is expressed with it Property.
Apply in vivo, pharmaceutical composition be the parenteral preferably applied (for example, intra-articular, intravenous, intraperitoneal, subcutaneously Or intramuscular).In certain embodiments, pharmaceutical composition is applied by bolus intravenous or intraperitoneal.Other route of administration bags Part (skin, eye, mucous membrane) is included, oral cavity, lung is intranasal, sublingual, rectum and vagina.
In one embodiment, the method that the present invention provides the expression of regulation target polynucleotide or polypeptide.These methods are led to Often include the lipid particle for the nucleic acid for being relevant to the expression that can adjust target polynucleotide or polypeptide for making the cells contacting present invention.Such as Used herein, term " regulation " censures the expression for changing target polynucleotide or polypeptide.Regulation can represent to increase or strengthen, or it can Represent reduction or reduce.
In related embodiment, the present invention provides the disease or disease for the overexpression that polypeptide is characterized as in treatment subject The method of disease, including pharmaceutical composition of the invention is provided to subject, wherein therapeutic agent is selected from siRNA, and microRNA, antisense is few Nucleotides, and the plasmid of siRNA, microRNA, or ASON, and wherein siRNA, microRNA or antisense RNA bag can be expressed Polynucleotides containing the polynucleotides for specifically binding coded polypeptide, or its complement.
In another further aspect, the present invention provides pharmaceutical composition, its lipid particle comprising the present invention and pharmaceutically acceptable Carrier or diluent.Representational pharmaceutically acceptable carrier or diluent include intravenous injection with solution (for example, salt solution or Dextrose).Composition can use creme, ointment, gel, the form of suspension or emulsion.
Following is representational LNP systems, the apparatus and method for manufacturing LNP systems, and uses the side of LNP delivering therapeutic agents The description of method.
Quick Microfluidic Mixing allows the generation of single dispersing lipid nanoparticle.The preparation of lipid nanoparticle is by setting Quick mixing lipid-ethanol solution is calculated as inducing within the microfluid mixer of mixed and disorderly advection with aqueous buffer to carry out (figure Reynolds numbers (24 15B) and in the middle of providing<Re<240) hybird environment of control.Microfluidic channel contains by change half The orientation of herringbone structure between circulation produces the herringbone part flowed in a jumble, causes the local cycle for rotating and extending flow center Property change.
In order to determine the mixed performance within device, using the pH sensitivity of fluorescein, wherein by 2 10 μM of fluorescein streams Mixing, a fluorescence is in pH8.88 and other non-fluorescence in pH5.15.Generation mixes (mixability>95%) passage needed Length is found to be between 0.8cm and 1.0cm.This causes to 0.1ml/min, 0.4ml/min, 0.7ml/min and 1.0ml/min The incorporation time of flow velocity, respectively about 45ms, 10ms and 5ms and 3ms.The small difference of expected mixing length, described in mixed and disorderly stream Mixed and disorderly stream only grown with P é clet number logarithms (Pe=Ul/D, wherein U are fluid velocities, and l is cross section passage length, And D is molecule diffusion).
The following preparation that represents is including ionizable cation lipid, DLin-KC2-DMA, with obvious 6.7 pKa, Cause lipid to be adapted to and siRNA is wrapped up with low pH, and provided in physiology pH close to neutral cation surface charge density.Use this LNP-siRNA schemes determine the effect that flow velocity is formed to LNP as model system.Due to increased flow velocity incorporation time It is substantially reduced, the speed of lipid importing aqueous phase, which is contemplated to, influences their final size and dispersiveness.Using same flow velocity, 0.1ml/min~1ml/min/ passages, Figure 16 B show the average grain of the LNP-siRNA systems produced by microfluid mixer Footpath.Buffer contains siRNA to produce 0.06 (wt/wt) siRNA/ total lipids ratio, and LNP mixtures are directly diluted into slow Electuary, to reduce ethanol content to about 22vol%.When increasing overall flow rate 0.2ml/min~2ml/min, particle diameter is significantly reduced. Particle diameter is maximum under 0.2ml/min flow velocity, and LNP reaches about 40nm arrowhead, such as by the particle size determination of number-weighting. Or, adjust incorporation time also by change ethanol and current ratio.It is effectively increased flow rate of water flow and provides lipid current more Fast dilution.As lipid-ethanol stream is held constant at 0.5ml/min, the increase of water flow velocity causes particle diameter to reduce (Figure 16 C).With The substantial reduction of 3- times of increased particle diameter of water flow velocity, about 70nm~35nm shows the important of quick reduction ethanol content Property.
Because these LNP are expected to spontaneously form and met as lipid to more aqueous environments, the effect for exploring lipid concentration is also weighed Will.As lipid concentration increases, it can be expected to increase or be additionally formed extra particle using to be merged into LNP lipid amount. Monitored, as the lipid concentration in ethanol stream from 10mM increases to 50mM.As the increase of lipid concentration is observed averagely Particle diameter about 40nm~70nm increase (Figure 17).
Quick Microfluidic Mixing provides the LNP-siRNA systems of wide preparation scope.With the improvement recently of cation lipid Several times of the LNP effect that has been in progress, also become obvious, further improve can the optimization through LNP compositions provide.Especially, it can Influence their bilayer-stabilization removal capacity and in vivo dissolving potentiality can influence their the circulation behavior in physiology pH. For example, the preparation with less PEG- lipids and increased cation lipid shows the internal of the LNP systems of hepatocytes-targeting Effect is significantly improved.This observes that it provides ED50 in the LNP optimized in the report recently of mouse factor VII models Further 5 times of reductions.Although PEG- lipids are required for particle stability, it can also reduce the film of these LNP systems-go Stabilize property.With pre-formed vesica (PFV) method, there is the LNP systems for being less than 5mol%PEG- lipids when attempting to produce When difficult met pair；This is probably due to increase less PEG content outside the vesica of the fusion between LNP.Moreover, pre-formed Lipid particle organize again incubation step necessary to the parcel with siRNA need 30% (v/v) scope ethanol solution.This Increased lipid fluidity can promote unstability and cause the extra aggregation and fusion of pre-formed lipid particle.
Using PEG-c-DMA, microfluid (MF) method is explored (fast mixed before the LNP dilutions of 25% ethanol (v/v) below Conjunction time and short resident) produce the abilities of the LNP-siRNA systems with the PEG- lipid contents changed.With 0.06 (wt/wt) SiRNA/ total lipids ratio use DLin-KC2-DMA, DSPC, cholesterine and PEG-c-DMA initial composition (40:11.5: 38.5:10mol/mol).Extra cholesterine is used for the PEG-c-DMA for compensating the amount of reduction.Use microfluidic methods PEG-c- DMA to 2mol% titration causes only a small amount of increase of particle diameter.Being further reduced to 1mol%PEG causes diameter about 20nm~about 40nm increase (Figure 18 A).On the contrary, shown using the average grain diameter of PFV methods as PEG- lipid contents are reduced to 1mol%, Particle diameter 20nm~70nm constant increase.In addition to producing the LNP of the PEG- lipids with low amounts, it is interesting can change it is positive from The amount of sub- lipid.As DLin-KC2-DMA from 40mol% increases to 70mol%, those produced by microfluidic methods are seen Observe the general increase (Figure 18 B) from about 40nm to 70nm of particle diameter.
The self assembly of microfluidic device can be produced with the close LNP completely wrapped up.Producing LNP-siRNA systems In, sane process will necessarily provide the high percent parcel of OGN products.By changing siRNA/ from 0.01 to 0.2 (wt/wt) Total lipid is than evaluating siRNA parcels using the LNP-siRNA preparations with 1mol%PEG.LNP preparations reach in this scope to be connect Nearly 100% percentage parcel (Figure 19).Reach 0.21 (wt/wt) siRNA/ total lipids ratio, corresponding to cation lipid and After charge balance (N/P=1) between anion siRNA, it was observed that parcel reduces (data are not shown).Trend is pre- after this Phase is due to compound siRNA and is wrapped in the not enough cationic charge needed in LNP.
Form visualizes the LNP produced by microfluid and pre-formed vesica method with freezing-TEM.LNP grain Footpath is similar to by dynamic light scattering measurement.Contain the siRNA with 0.06wt/wt:Lipid than 40/11.5/47.5/ 1mol% DLin-KC2-DMA/DSPC/ cholesterine/PEG-c-DOMG LNP-siRNA systems are shown in Figure 20 A.In addition, phase The empty LNP samples of same composition are shown in Figure 20 B.The particle of generation is dimensionally mainly spherical and homogeneous.Also will be with advance The LNP imagings of composition that the method for formation is prepared and same.These share similar characteristics with microfluid LNP, although also observing Other features such as coffee-beans structure.These LNP sizes are also bigger, as from dynamic light scattering outcome expectancy.
The LNP siRNA systems produced by Microfluidics can be the strong internal gene silencing agent of height.Use mouse After factor Ⅴ II scale-model investigations intravenous injection in LNP siRNA system induction bodies gene silencing ability.Use microfluidic methods Create containing the siRNA with 0.06 (w/w):Lipid than DLin-KC2-DMA/DSPC/ cholesterine/PEG-c-DOMG system Agent.LNP-siRNA is applied by tail vein injection.Cation lipid, DLin-KC2-DMA is changed for 30mol%~60mol% And keep 0.2wt/wt DSPC:Cholesterine ratio.Cation lipid content in increase LNP causes the progressive of FVII silences to change It is kind.The LNP of optimal representation contains 60mol%DLin-KC2-DMA, causes the effective agent of about 0.03mg/kg 50%FVII silences Measure (Figure 21).With interest it is noted that being further increased to 70mol% causes without more than 60mol%DLin-KC2-DMA LNP Effect observable improvement.
As a result show, the microfluidic device containing herringbone blender staggeredly, which can be used for producing, has the combination of various lipids The LNP of thing, available for effective parcel OGN such as siRNA, and displaying, the LNP siRNA systems of generation present it is excellent external and Internal gene silencing ability.
The Microfluidics device and system of the present invention allow the LNP to form LNP and the OGN containing 100nm sizes or smaller And OGN parcels 100% are provided.On LNP formation, the speed of mixing and be important parameter than substantially.Ethanol-lipid soln with The quick mixing of aqueous buffer causes the increased polarity of medium, and it reduces the solubility of the lipid of dissolving, causes them to sink Form sediment solution and formation nano-particle.Quick mixing causes solution to be rapidly achieved the equal body of lipid exceeding through whole mixed volume Saturation state, causing the quick and homogeneous of nano-particle has coring.The increased of nano-particle has coring and growth to exhaust surrounding The free lipid of liquid, from there through the subsequent growth of aggregation limitation of free lipid.The mechanism of this suggestion is consistent with following observation： The lipid (the free lipid of reduction) of lower concentration causes smaller LNP (see Figure 17) in ethanol, and higher flow rate causes to satiety The faster and more homogenizing method of sum, causes smaller LNP formation, and increase aqueous solvent to compare organic solvent constituent Also more small particles (Figure 17) are caused.
The OGN parcel efficiency close to 100% is presented in the LNP OGN systems of the invention prepared by Microfluidics method. The presence of small multi-layer vesicles is disclosed using freezing-TEM researchs before the PFV technologies for antisense OGN, causes following possibility Property, parcel is related to be adsorbed to the OGN of pre-formed vesica, and it is associated as with the extra pre-formed vesica around former vesica Have nucleation site.On the contrary, the LNP OGN produced by Microfluidics method the most of LNP systems of freezing-TEM studies have shown thats It is " entity core " structure and prompting, the different mechanism of OGN parcels are operational.Especially, these structures are existed with siRNA Assemble before nano-particle assembling or with nano-particle and associated simultaneously with cation lipid monomer unanimously.Microfluidics method is only Stand on nucleic acid composition and aid in antisense and siRNA OGN to be to surpass the side that reports before close to the ability of 100% parcel efficiency The main advantage of method.
Microfluidics method provides the advantage of the LNP synthetic technology alternative more than 3 kinds, including is typically used for producing LNP extrusion, pre-formed vesica method, and the spontaneous vesica forming method wrapped up for OGN.Microfluidics method is carried LNP for 100nm size ranges or smaller and, in the presence of cation lipid, it is allowed to which LNP is with low-level stabilisation PEG- fat Matter is formed.The shortcoming of Microfluidics method is related to the demand of removal ethanol after preparation, and specific lipid is relatively not in ethanol Dissolubility, and potential scaleability problem.Microfluidics method provides the advantage of parcel efficiency, is used using PFV processes hardly possible The use of high-cation lipid content and low PEG- lipid levels, the removal of the demand to producing pre-formed vesica, and use As little as 150 μ g oligonucleotides, the ability criticized on a small scale is produced due to few loss of the small dead volume (1 μ l) of equipment.
Compared to SVF " T pipes " process for producing the LNP systems for loading OGN, the advantage of Microfluidics method is similar to Those of PFV processes instruction, in addition to not needing pre-formed vesica.The aperture of T pipes is about 1.5mm diameters, it is necessary to high stream Speed (>1ml/s), occurs the quick speed for mixing needs to reach.Micro-mixer allows LNP OGN preparations well defining, Occurring due to the loss of dead volume with much lower flow velocity and reduction under the conditions of reproducible, it is allowed to for LNP optimizations With the more directly preparation criticized on a small scale of testing in vitro.
LNP OGN system scalabilities.Although the device of the Peak Flow Rate with 1ml/min can be not enough, single Microfluidics core Piece can be containing 10 or more micro-mixers, to reach about 10mL/min overall flow rate.It is assumed that the relatively inexpensive property of this technology, Being used in parallel many chips can put into practice, and potentially allow for the flow velocity from single desk-top instrument 100ml/min or higher.This Outside, upstream fluid operation easily can be incorporated into the device, can close to allow from the accurate programmable preparation of multiple components Into preparation and feature highly advantageous in the screening and optimization of parameter.
【Entity core LNP】
Bilayer vesicle structures of the specific LNP siRNA preparations model prompting siRNA in the LNP of the inner side of aqueous interior. But many observation promptings, the model is incorrect, at least for by LNP siRNA systems that Microfluidic Mixing method is produced Speech.For example, the freezing of the LNP siRNA systems produced by Microfluidic Mixing-Electron microscopy indicates there is electron dense Core, rather than the aqueous core consistent with imitated vesicle structure.As described above, the preparation of LNP siRNA systems can routinely cause to approach 100% siRNA parcel efficiency, is the observation inconsistent with the double-decker of wherein expectable 50% maximum parcel efficiency.
Using various physics and the structure of enzymatic evaluation of measuring LNP siRNA systems.The result instruction of acquisition, these LNP SiRNA systems are with the entity core inner by the siRNA monomer compositions being combined with cation lipid and organize into inverted The lipid of micelle or the structure of correlation.
LNP systems are presented in the presence and absence of the siRNA of parcel by freezing the electron dense entity core that EM is indicated Core structure.The LNP systems produced by Microfluidic Mixing are presented such as by freezing the visual electron-dense cores of EM, with entity Core is consistent, and the aqueous core texture of the LNP siRNA system recommendations with being created by alternative method is opposite.This confirms such as Just by containing siRNA DLin-KC2-DMA/DSPC/Chol/PEG- lipids (40/ with 0.06siRNA/ lipids (wt/wt) content 11.5/47.5/1；Mol/mol) composition LNP siRNA preparations be shown in Figure 22 A, its correspond to 4 negative electrical charge (in siRNA On) to positive charge (on the cation lipid protonated completely) N/P ratios.As a result about 75% cation lipid is not compound to SiRNA in LNP.Entity core electron compact texture (figure in contrast with by the deficient close inside of the POPC Vesicular systems constituted 22B), and inside visually similar POPC/ trioleins (POPC/TO) LNP electron dense (Figure 22 C).Pass through Microfluidic Mixing The POPC/TO LNP of generation are made up of the TO surrounded by POPC individual layer hydrophobic core.
The interested of Figure 22 A is characterized in that 75% ionizable cation lipid is not compound in siRNA, but LNP Entity core inner is presented in siRNA particles entirety.This prompting, cation lipid can contribute to entity core inner, or even when it When not being compound in siRNA.The LNP systems constituted with identical lipid but without siRNA are prepared using Microfluidics process, and it is logical Cross freezing EM signs.As depicted in Figure 22 B, siRNA missing is observed in electron-dense cores, is indicated, ionizable sun Cationic lipid such as DLin-KC2-DMA, may be combined with DSPC and cholesterine, and non-lamelliform electronics can be used inside LNP Compact texture.
LNP structures are presented arrowhead and indicated, ionizable cation lipid is internally formed inverted micelle knot in LNP Structure.The contribution of cationic lipid confrontation electron dense LNP cores lift the LNP systems can why molecular structure the problem of.Thus Reasonable proposal, the cation lipid associated with to ion uses inverted structure, such as inverted micelle, pair with these lipids In the inverted structure such as hexagon H in the mixture with anion lipidIIThe tendency of phase is consistent.And then, this can be pointed out, by The arrowhead of the diameter in the range of 10nm should be presented in the LNP systems of net cation lipid composition, and it, which is substantially surrounded, has 2 double-deck thickness inside 2~3nm of diameter inverted micelle.HIIThe aqueous channels that phosphatidyl-ethanolamine is found in phase Diameter is 2.6nm.Microfluidics production process provides the fast hybrid power that driving is produced to the arrowhead system of LNP systems Learn.Evaluate the arrowhead that can be reached to DLin-KC2-DMA/PEG- lipid systems (90/10, mol/mol).As depicted in figure 23, confirm that particle diameter is being diametrically about by the measurement of the dynamic light scattering to these LNP formed by microfluidic methods 10nm, has found inconsistent with significantly aqueous core or the volume of capture.
Related the problem of, concerns the structure of cation lipid-siRNA compounds.Again, reasonably speculate, its by around The inverted micelle composition of the distortion of the cation lipid of siRNA oligonucleotides.And then, this can point out 15~20nm scope Arrowhead, estimates the siRNA contained in this inverted micelle and is surrounded by the inside individual layer of cation lipid, then by remaining fat The external mono-layers of matter are surrounded, and siRNA yardstick is 2.6nm diameters and 4.8nm length.In order to determine whether that this is consistent with experiment, The LNP siRNA systems of arrowhead by with the N/P corresponding to measure than high-caliber siRNA DLin-KC2-DMA and PEG- lipids (90/10；Mol/mol) constitute.As depicted in Figure 23, siRNA include cause the arrowhead of about 21nm diameters System, this is consistent with hypothesis.
The siRNA of parcel is fixed in LNP.If the entity that siRNA is combined and is positioned within LNP with cation lipid Core, if can be expected compared to deficient movement of freely collapsing in the aqueous interior of bilayer vesicle system.SiRNA motility can Use31P NMR technologies are detected.Especially, the motion that limitation can be expected averagely can be for compound siRNA it is possible, due to The big chemical shift anisotropy of phosphate, causes very wide " solid state "31P NMR resonate., should under conditions of use Resonance will not be detectable.If on the other hand, siRNA can freely collapse in aqueous environments, the quick average meeting of motion It is expected that cause it is narrow, easily it is detectable,31P H NMR spectroscopies.Occur certainly occurring from phosphatide phosphorus to eliminate31P NMR signals and It was found that as ignoring from LNP preparations DSPC, to test this hypothesis.As depicted in Figure 24 A, for constituting DLin-KC2- with lipid DMA/Chol/PEG- lipids (50/45/5mol%) and contain siRNA (0.06siRNA/ lipids；Wt/wt LNP siRNA systems) System, does not observe to the siRNA of parcel31P NMR signals, it is consistent with the fixation within LNP cores.If by detergent sodium dodecyl Base sodium sulphate adds (1%) to dissolve LNP and release parcel siRNA, then detects narrow31P NMR signals, as depicted in figure 24C。
The siRNA of parcel is fully protected from being degraded by outside RNaseA.The test of siRNA internalization is, if SiRNA completely cuts off in LNP cores, and they should be fully protected from being degraded by the RNase of outside addition.There to be lipid composition The LNP siRNA systems of DLin-KC2-DMA/DSPC/Chol/PEG- lipids (40/11/44/5mol%) are incubated with RNaseA, It can be digested with the siRNA for determining whether parcel.As depicted in the gel provided in Figure 25, free siRNA degrades, and siRNA What is manufactured within the LNP particles of association is protected (Figure 25 arrow marks) completely by microfluidic methods.Such as it is also shown in Figure 25, decontamination Agent Triton X-100, which are added to LNP, causes LNP dissolving, siRNA release, and the degraded in the presence of RNase.
The siRNA of parcel and the cation lipid of internalization are compound.The entity core of LNP siRNA systems by with cationic lipid The siRNA compositions of the compound parcel of matter, and remaining lipid (cation lipid, cholesterine and PEG- lipids) be present in it is inverted In core in micelle or similar structures, or outside LNP.For high siRNA contents, wherein substantially all of cationic lipid The siRNA of matter and internalization is combined, and can be expected, and few cation lipid can be positioned at outside LNP.Develop FRET (FRET) determine to determine external cationic lipid.Determining needs to prepare by containing FRET pairings with high (itself-quenching) concentration, The electronegative vesica LNP of NBD-PE/Rh-PE dioleoyl phosphatidylserine (DOPS) composition.Then will be electronegative DOPS LNP and it is made up of pH5.5 DLin-KC2-DMA/DSPC/Chol/PEG- lipids (40/11.5/47.5/1mol%) LNP siRNA systems are incubated.DLin-KC2-DMA pKa is 6.7, thus, the almost all DLin-KC2-DMA of LNP outside Can be powered in pH5.5, promote the interaction with electronegative DOPS LNP and potentially merge.Fusion is reported as in 535nm After the increase of the NBD-PE fluorescence at place, such as lipid mixing, NBD-PE and Rh-PE probes become dilution.
As depicted in Figure 26, when LNP systems do not contain siRNA, it was observed that Substantial Merging and significant ratio The DLin-KC2-DMA of the external mono-layers of LNP systems is consistent.But, when LNP systems with 0.06 (wt/wt) siRNA to lipid Than when its just (cation lipid) electric charge for corresponding to 4 contains siRNA to negative (siRNA) N/P charge ratios, fusion is substantially reduced (Figure 26), however, for 1 N/P preparations LNP siRNA systems, it was observed that less or amixis, indicating, less or without DLin- KC2-DMA is present in outside LNP siRNA.This supports hypothesis, high siRNA contents, substantially all cation lipid and siRNA It is compound, and completely cut off inside LNP.
As a result evidence is provided, the inside of LNP siRNA systems is by the siRNA monomer compositions by being combined with cation lipid Entity core, and constituted with inverted micelle or the lipid of the structural arrangement of correlation.These results imply LNP siRNA structures Model produce LNP with for the appropriate property of application-specific there is provided the principle of accessible high siRNA parcel efficiency and prompting The method of siRNA systems.
The model of LNP siRNA structures based on result is shown in Figure 27.Model advises, the siRNA of parcel by sun from In the inverted micelle for the distortion that sub- lipid is surrounded, and remaining lipid tissue enters in the inverted micelle around anion to ion Also outermost individual layer is constituted.
How model can reach during Microfluidic Mixing production process close to 100% siRNA parcel efficiency if being provided Understand.This is the subject matter that siRNA is wrapped up in two-layer system, because presumption cation lipid is equally distributed in bilayer Both sides, can be expected maximum 50%siRNA internalizations.Model points to LNP siRNA sizes, and composition and surface charge are easily adjustable Mode.On size, arrowhead structure is substantially the structure containing a siRNA monomers/particle, points out about 15~20nm's Arrowhead.The LNP siRNA particles are easily reached using the microfluidic methods of the present invention.By the limit of siRNA monomer composition Size LNP siRNA systems processed potentially can be used as component using Microfluidic Mixing technology, and composition and surface electricity are changed to reach The LNP siRNA systems of lotus.Pre-formed arrowhead LNP siRNA are quick with the ethanol solution containing electronegative lipid Mixing, for example, it is contemplated that cause to interact with excess cations lipid to produce internal inverted micelle core texture and band The surface of negative electricity.
The lipid particle of invention as described herein includes the component of (that is, comprising) narration.In certain embodiments, originally The particle of invention includes the component of narration and does not influence the feature of particle (that is, the particle being substantially made up of the component described) Other extra components.Influence particle ' feature extra component include following component, such as inadvertently change or influence particle Non-therapeutic aspects and effect extra therapeutic agent, inadvertently change or influence particle dissolving narration therapeutic ingredients The extra component of ability, and inadvertently change or influence particle increase narration therapeutic ingredients bioavilability energy The extra component of power.In other embodiments, particle of the invention includes the component (that is, being made from it) only described.
The following example is provided with purpose of illustration, it is not limited, it is desirable to the invention of protection.
【Embodiment】
【Material】
DOPC (DOPC), 1,2- distearyl-sn- glyceryl -3- phosphoric acid courages Alkali (DSPC), 1,2- dioleoyl-sn- glyceryl -3- phosphoserines (DOPS), 1,2- dioleoyl-sn- glyceryl -3- phosphoric acid Ethanol amine-n-(7- nitros -2-1,3- benzene and oxadiazole -4- bases) (NBD-PE), 1,2- dioleoyl-sn- glyceryl -3- phosphoric acid second Alcohol amine-n-(lissamine rhodamine B sulfonyls) (Rh-PE) is obtained from Avanti Polar Lipids (Alabaster, AL).4- (2- ethoxys) piperazine -1- ethyl sulfonic acids (HEPES) and cholesterine are obtained from Sigma (St Louis, MO).N- [(poly- (the second of methoxyl group Glycol)2000) carbamoyl] -1,2- myristyl epoxide propyl group -3- amine (PEG-C-DMA) is by AlCana Technologies is synthesized.2- (N- morpholinoes) ethyl sulfonic acids (MES) are obtained from BDH.Ammonium acetate, sodium acetate and sodium chloride are obtained from Fisher Scientific(Fair Lawn,NJ).RNaseA be obtained from Applied Biosystems/Ambion (Austin, TX).Factor Ⅴ II (FVII) is targetted, and low GC negative controls siRNA is purchased from Invitrogen (Carlsbad, CA).Factor Ⅴ II siRNA：(SEQ ID NO:1) 5 '-GGAUCAUCUCAAGUCUUACTT-3 ' (FVII justice), and (SEQ ID NO:2)5’- GUAAGACUUGAGAUGAUCCTT-3 ' (FVII antisenses).DLin-KC2-DMA is obtained from AlCana Technologies Inc. (Vancouver,BC)。
【Embodiment 1：The preparation of LNP systems：Pre-formed vesica method】
In the present embodiment, the preparation of the LNP-siRNA systems using pre-formed vesica method is described.
Using being such as depicted in Figure 15 A and be such as described in N.Maurer, K.F.Wong, H.Stark, L.Louie, D.McIntosh,T.Wong,P.Scherrer,S.Semple and P.R.Cullis,Spontaneous Entrapment of Polynucleotides Upon Electrostatic Interaction With Ethanol Destabilized Cationic Liposomes:Formation of Small Multilamellar Liposomes,Biophys.J.；80: 2310-2326 (2001) pre-formed vesica method manufacture LNP-siRNA systems.By cation lipid, DSPC, cholesterine and PEG- lipids are dissolved in ethanol with appropriate mol ratio first.Then lipid mixture is added drop-wise to aqueous buffer (citric acid Salt or acetate buffer, pH4), while being vortexed to 30% (v/v) final ethanol and lipid concentration.Then by the fat of hydration Matter uses Lipex extruders (Northern by the filter (Nuclepore) of 80nm holes-size of 2 stackings in room temperature Lipids, Vancouver, Canada) extrude 5 times.SiRNA (being dissolved in the same aqueous solution containing 30% ethanol) is added into capsule Steep suspension and mix.Usually using 0.06 (wt/wt) target siRNA/ lipid ratios.This mixture is incubated 30 minutes in 35 DEG C See, to allow vesica to organize to wrap up with siRNA again.Then ethanol is removed, and by external buffer agent with phosphate buffer (PBS) By dialysing, (12-14k MW cutoffs, Spectrum medical instruments) substitutes onto 50mM Citrate buffers Then agent, pH4.0 carries out dialysis PBS, pH7.4.
【Embodiment 2：The preparation of LNP systems：The herringbone blender that microfluid interlocks】
In the present embodiment, the representative LNP- of the invention of the herringbone blender interlocked using microfluid is described SiRNA systems.
It is prepared by LNP-siRNA.Oligonucleotides (siRNA) solution is prepared in pH4.0 25mM acetate buffers.Rely on In desired oligonucleotides:Lipid ratio and formulation concentrations, solution is prepared with the target level of 0.3mg/ml~1.9mg/ml total lipids. Prepare in ethanol lipid soln that DLin-KC2-DMA, DSPC, cholesterine, and PEG- lipids are contained with appropriate mol ratio and The concentration of alcohol for reaching 90% (v/v) is diluted to 25mM acetate buffers.Figure 15 B are the microfluidic devices used in this Schematic diagram.Device has 2 entrances, and one is used for each solution made above, and one outlet.Microfluidic device passes through soft The duplicating molded of the motherboard of micro- construction is produced in photoetching, elastomer.Device characteristic is 200 μm wide and 79 μm high, with feature For the hybrid channel for being formed as 31 μm high and 50 μ m-thicks herringbone structures on passage top.Fluidly connecting is used for attaching to I.D., 3/32 " O.D. pipelines are carried out the 1/32 of the 21G1 pins being connected with syringe ".1ml syringes are generally used for 2 entrance streams. Double syringe pump (KD200, KD science) is used to control the flow velocity by device.The flow velocity that respectively flows change for 0.1ml/min~ 1ml/min.2 kinds of solution are imported microfluidic device by syringe pump, and (entrance a and entrance b) in Figure 15 B, wherein they are in Y- companies Contact.Occur inapparent mixing by the diffusion put at this under laminar flow, but 2 kinds of solution pass through herringbone with them Structure and mix.
The mixing occurred in these structures by mixed and disorderly advection, causes the characteristic separation that lamination is flowed to become less and less, Thus quick diffusion is promoted.This mixing occurs with ms time scales, and causes lipid to be transferred to progressively more aqueous environments, reduces Their solubility and cause spontaneously forming for LNP.By including cation lipid in lipid composition, pass through positively charged What the associating of lipid head group and electronegative oligonucleotides obtained oligonucleotides material obtains collection.Mix it in microfluidic devices Afterwards, LNP mixtures are generally diluted into the vial of the buffer of the stirring containing 2 volumes.By carrying out dialysis 50mM lemons Then hydrochlorate buffer, pH4.0 carries out dialysis PBS, and pH7.4 finally removes ethanol.The oligonucleotides lacked from cushioning liquid As produce empty pocket bubble.
LNP graphical analyses is measured during mixing by the fluorescence imaging of the mixing of the luciferin solution with different pH value Between.Kalman filtered models using the inverted Laser Scanning Confocal Microscopes of Olympus using 10 × object lens and with 2 scannings per line are received Collect picture.25 pieces being equally spaced are taken along channel height, and combine to determine overall strength feature.For the position of each imaging, 10 adjacent lines along the pixel in stream direction are averaged, to obtain the strength characteristic along channel width, and it is mixed to determine Conjunction degree.Combined experiments with supplement 0.5M NaCl 2 10 μM of luciferin solutions carry out, with due to sodium and phosphate anion it is dense Degree big difference and suppress liquid connect gesture formation.A kind of solution contains 14mM PBs, pH8.88, and other contain There are 1mM PBs, pH5.15.Originally the small of fluorescence in alkaline solution can be overwhelmed in the increase of pH5.15 solution fluorescence Reduction, causes total fluorescence intensity to increase by 2 times.Mixability uses 0.1ml/min, 0.4ml/min, 0.7ml/ along passage length The measurement of rate of flow of min and 1.0ml/min individual flow is about 2.1mm, 6.2mm and 10.1mm.
LNP characterizes particle diameters and uses the Submicron Particle Sizer of Nicomp models 370 by dynamic light scattering (Particle Sizing Systems, Santa Barbara, CA) is determined.Use number-weighting and intensity-weighting distribution Data.Determine to measure total cholesteric by using the cholesterine E enzymatics from Wako Chemicals USA (Richmond, VA) Alcohol confirms lipid concentration.Free siRNA removal VivaPureD MiniH posts (Sartorius Stedim Biotech GmbH, Goettingen, Germany) carry out.Then crack eluent in 75% ethanol, and by siRNA by Absorbance is measured at 260nm to quantify.By oligonucleotides before free oligonucleotides content is removed and afterwards than determining Efficiency is wrapped up, lipid content is standardized as.
LNP Cyro- transmit Electron microscopy by by the 3 μ L LNP containing 20~40mg/ml total lipids PBS Sample is prepared applied to the standard electronic microscope inspection grid of the carbon film with perforation.By using Vitrobot systems (FEI, Hillsboro, OR) trace then in liquid ethane plug-freezing LNP suspension and in the film of amorphous ice snap frozen Vesica removes excess liq.Under cryogenic, with 29K magnifying power, camera is installed with AMT HR CCD sides and imaged. 70 degree of freezings of Gatan-transfer support in FEI G20 Lab6 200kV TEM, it is weak with 5~8 μm under the conditions of low dosage Jiao loads sample, to strengthen picture contrast.
For the LNP-siRNA of the FVII activity week old of activity in vivo .6~8, female C57Bl/6 mouse are obtained from Charles River Laboratories.LNP-siRNA containing factor Ⅴ II siRNA is filtered by 0.2 μm of filter and it is being used The preceding concentration that needs are diluted in sterile phosphate buffer.Through lateral tail vein, made with intravenous apply of 10ml/kg volume Agent.After 24h, animal ketamine // Xylazine anesthesia, and blood is collected by cardiac puncture.Sample is processed as serum (Microtainer Serum Separator Tubes；Becton Dickinson, NJ), and -70 are tested or are stored in immediately DEG C it is used for the analysis of hereafter serum factor VII levels.According to Institutional Animal Care and Use Committee (IACUC) is applicable and area of approval, and state and federal regulation carry out all processes.
Serum factor VII levels are determined using colorimetric Biophen VII calibrating kits (Anaira).Control serum is closed And and serial dilution (200%~3.125%) with produce be used for calculate processing animal FVII levels calibration curve.Use Biophen VII kits analyze the plasma sample (n of the appropriate dilution of the animal from processing according to the operation instruction of manufacturer =3 per dosage) and saline control group (n=4).It is flat in 96- holes, non-combination polystyrene assay plate (Corning, Corning, NY) in analyzed, and at 405nm measurement absorbance.By the calibration produced with the control serum of serial dilution Factor Ⅴ II levels in the animal of curve determination processing.
【Embodiment 3：LNP systems：Entity core】
In the present embodiment, the structure of the representative LNP-siRNA systems with entity core of the present invention is described.
Lipid nanoparticle prepares by the way that the dense storing solution of lipid in the ethanol of desired volume and aqueous phase are used Above-mentioned micro--blender mixes to prepare LNP.For siRNA parcel, it would be desirable to amount siRNA and 25mM acetate buffer Agent, pH4 mixing.The lipid of equating volume/ethanol phase and siRNA/ aqueous phases are closed with micro--blender containing herringbone structure And, to promote mixing.After micro--blender is removed by ethanol content sodium acetate buffer rapid dilution be 25%.By it is micro-- The flow velocity of mixing is regulated and controled using double-syringe pump (Kd Scientific).Lipid mixture is then in 50mM MES/ citric acids Experience is dialysed for 4 hours in sodium buffer (pH6.7), is the dialyzed overnight in phosphate buffer (pH7.4) afterwards.
Freezing-EM. by the 3 μ L LNP containing 20~40mg/ml total lipids PBS by being applied to the carbon with perforation The standard electronic microscope inspection grid of film prepares sample.By using Vitrobot systems (FEI, Hillsboro, OR) trace, Then in liquid ethane plug-freezing LNP suspension and amorphous, snap frozen vesica was removed in vitreum ina thin film Quantity of fluid.Under cryogenic, with 29K magnifying power, camera is installed with AMT HR CCD sides and imaged.Use FEI G20Lab6 The degree of Gatan 70 in 200kV TEM freezes-shift support, under the conditions of low dosage, with 5~8 μm it is weak it is burnt load sample, With Enhanced Imaging contrast.
RNase protection determine by the factor Ⅴ II siRNA cholesterine of 40%DLinKC2-DMA, 11%DSPC, 44% and 5%PEG-c-DMA is wrapped up using Microfluidics mixed method.By 1 μ g siRNA in 50 μ L 20mM HEPES (pH7.0), Incubated 1 hour in 37 DEG C with 0.05 μ g RNaseAs (Ambion, Austin, TX).Ended incubating, by the reaction of 10 μ L aliquots Mixture is added to 30 μ L FA dyestuffs (formamide of deionization, TBE, PBS, xylene blue, bromophenol blue, yeast tRNA), with Suspend RNase reaction.Gel electrophoresis is carried out using 20% native polyacrylamide gel, and by nucleic acid by using CYBR-SAFE (Invitrogen, Carlsbad, CA) dyeing visualization.
31P-NMR researchs uses the spectrometers of Bruker AVII 400 operated with 162MHz to obtain what proton-decoupled joined31P H NMR spectroscopy.Corresponding to about 104The free induction decay (FID) of scanning is with 15 μ s, 55- degree pulses, 1s interpulse delays and 64kHz Spectral width obtain.The index multiplication corresponding to 50Hz line width is applied to FID before Fourier conversions.Use The temperature units of Bruker BVT 3200 regulate and control sample temperature.Measured in 25 DEG C.
FRET film control fusion examine and determine LNP siRNA by using the lipid mixed determining of FRET Fusion between nano-particle and anion DOPS vesicas.By using the appropriate direct rehydration lipid film of buffer, Zhi Houshi Preparation is extruded for 10 times using Lipex extruders by 100nm apertures polycarbonate membrane and contains NBD-PE and Rh-PE (1mol Each %) mark DOPS vesicas.With 0,0.06 and 0.24 siRNA:Prepared by lipid ratio (D/L ratios, wt/wt) includes 40% DLinKC2-DMA, 11.5%DSPC, 47.5% cholesterine, 1%PEG-c-DMA LNP.D/L=0.24 represent equimolar than Just (cation lipid) is to bearing (siRNA) electric charge (N/P=1).Carry out lipid combined experiments.Do not mark the DOPS vesicas of mark and The LNP of note is with 1:2 mixed in molar ratio enter the 10mM acetates containing 2mL, and 10mM MES, 10mM HEPES, 130mM NaCl are put down Weigh to pH5.5 stirring cuvette.Excited using 465nm, and 535nm transmittings are made using LS-55 Perkin Elmer fluorescence photometers With 1 × 1cm cuvettes under continuous stirring at low speed, NBD-PE fluorescence is monitored.Lipid is mixed into monitoring about 10min, after it 20 μ L 10%Triton X-100 are added, to destroy whole lipid vesicles, infinite probe dilution are represented.Use following equations Determine and mixed as the lipid of the percentage of infinite probe dilution：% lipids mixing=(F-Fo)/(Fmax-Fo) x100, wherein F is Fluorescence intensity during measure at 535nm, FoIt is initial fluorescent intensity, and FmaxIt is unlimited after addition Triton X-100 The maximum fluorescence intensity of probe dilution.
【Embodiment 4：The continuous assembling of lipid nanoparticle】
In this instance, the representative method of the present invention, the continuous assembling for manufacturing lipid nanoparticle are described.
Oligonucleotides (siRNA) solution is prepared with 1.31mg/ml in 25mM acetate buffers, pH4.0.Lipid is mixed Thing is prepared into containing 90mol% cation lipids (DLin-KC2-DMA) and 10mol%PEG-c-DMA (10mM total lipids dissolvings In ethanol).2 kinds of solution are mixed and used 25mM acetate buffers with overall flow rate 2ml/min using microfluid mixer, PH4.0,2 times of dilution forms initial or core nanoparticles to cause ethanol to drop to about 23vol%.By taking this initial lipid Particle suspension, by itself and another fat containing the anion lipid dioleoyl phosphatidylserine (DOPS) for being dissolved in methanol Matter solution is mixed, and is also diluted to about 25vol% solvents (methanol and ethanol) continuously to be assembled.With to cation lipid about 4 × molar excess adds the 2nd lipid, DOPS.Continuously assembled by being alternately repeated between cation lipid and anion lipid Journey.
Particle diameter uses Malvern Zetasizer Nano-ZS (Malvern Instruments by dynamic light scattering Ltd, Malvern, Worcestershire, UK) determine.Use the distributed data of number-weighting.Use Malvern Zetasizer The surface electricity of LNP systems is provided using capillary pond (DTS1060, the Malvern Instruments Ltd.) measurement that can be arranged The zeta potential measured of lotus.LNP systems are diluted to about 0.3mg/ml total lipids in 25mM acetate buffers, pH4.0.
【Embodiment 5：Represent preparation and the sign of lipid particle】
In this instance, describe only by cation lipid and nucleic acid (DLin-KC2-DMA-siRNA) constitute it is of the invention Represent lipid particle.
SiRNA solution is prepared with 0.38mg/ml in 25mM acetate buffers, pH4.0.Lipid soln is prepared into second DLin-KC2-DMA is contained with 10mM concentration in alcohol.siRNA:Lipid ratio is 0.06 (wt/wt).By each solution with equivalent flow velocity Microfluid mixer is inputted with 2ml/min overall flow rate.By sample 25mM acetate buffers, pH4.0 further dilutes, made Ethanol content is 25vol%.
Particle diameter uses the Submicron Particle Sizer (Particle of Nicomp models 370 by dynamic light scattering Sizing Systems, Santa Barbara, CA, USA) determine.The progress sample measurement in 25mM acetates, and use number- The distributed data of weighting.Particle has 14.2nm average grain diameter, and the coefficient of variation is 0.487, and χ2It is 1.93.
Illustration and illustrated embodiment is described, those skilled in the art can agree to, various change can be carried out without de- From the spirit and scope of the present invention.
Other embodiments：
1. the method for the lipid particle containing nucleic acid is manufactured, including：
(a) the 1st conductance for including nucleic acid in the 1st solvent is entered into microfluidic device；Wherein device, which has, is suitable to one Or more the 1st area of conductance inflow device and for the 2nd area of the content that one or more streams are mixed with microfluid mixer；
(b) the 2nd comprising lipid particle-forming material in the 2nd solvent is flowed gatherer to provide in laminar flow condition 1st and the 2nd stream of lower flowing, wherein lipid particle-forming material include cation lipid, and wherein the 1st and the 2nd solvent not phase Together；
(c) one or more 1st streams and one or more 2 streams are made from the 2nd area of the 1st area's inflow device of device；With And
(d) one or more 1st streams are mixed in the 2nd area of device and one or more 2 streams include lipid to provide 3rd stream of particle, the lipid particle is included：
(i) core being made up of nucleic acid and cation lipid；With
(ii) the 2nd lipid of the core is surrounded；
Wherein due to the nucleic acid, the 31P of the lipid particle measured in the solution after being handled with 150mM ammonium acetates Resonance is not presented for nuclear magnetic resoance spectrum.
2. the method for embodiment 1, wherein the particle comprising nucleic acid, cation lipid, neutral lipid, PEG- lipids and Sterol.
3. the method for embodiment 1, wherein the cation lipid is ionizable lipid.
4. the method for embodiment 1, wherein the cation lipid is amino lipids.
5. the method for embodiment 1, wherein the 2nd lipid is selected from：Neutral lipid, anion lipid, and PEG- lipids, wherein The PEG- lipids are selected from：The phosphatidyl-ethanolamine of PEG- modifications, the phosphatidic acid of PEG- modifications, the ceramide of PEG- modifications, The dialkylamine of PEG- modifications, the diacylglycerol of PEG- modifications, the dialkyl glycerol of PEG- modifications.
6. the method for embodiment 1, wherein the nucleic acid is DNA, RNA, lock nucleic acid, nucleic acid analog or can express DNA Or RNA plasmid.
7. the method for embodiment 1, wherein the nucleic acid is mRNA, siRNA or microRNA.
8. a kind of microfluidic device is used for the purposes for manufacturing the nano-particle comprising core, the microfluidic device includes：
(a) the 1st entrance, it is used to receive the 1st solution；
(b) the 1st passage, it circulates with the 1st entrance is connected to provide the 1st stream of the 1st solution；
(c) the 2nd entrance, it is used to receive the 2nd solution；
(d) the 2nd passage, it circulates with the 2nd entrance is connected to provide the 2nd stream of the 2nd solution；And
(e) the 3rd passage, it is used to receive the 1st and the 2nd stream, is suitable to the 1st wherein the 3rd passage has under laminar flow condition The 1st area of the 3rd passage is flowed into the 2nd conductance and molten comprising the 1st to provide suitable for the 2nd area of the content of the stream of mixing the 1st and the 2nd 3rd stream of the mixture of liquid and the 2nd solution.
9. a kind of microfluid system is used for the purposes for manufacturing the nano-particle comprising core, the microfluid system includes：
At least one microfluidic device defined according to embodiment 8；
It is configured to supply the 1st pump of the 1st solution to the 1st entrance；With
It is configured to supply the 2nd pump of the 2nd solution to the 2nd entrance.
Sequence table
<110> The University of British Columbia
Cullis, P.R.
Belliveau, N.M.
Hansen, C.L.
Huft, J.
<120>Lipid particle and related methods containing nucleic acid
<130> UOBC-1-35680
<150> US 61/280510
<151> 2009-11-04
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>Synthesis
<220>
<221> misc_feature
<222> (1)..(21)
<223>Sequence is DNA/RNA crossbreds
<400> 1
ggaucaucuc aagucuuact t 21
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>Synthesis
<220>
<221> misc_feature
<222> (1)..(21)
<223>Sequence is DNA/RNA crossbreds
<400> 2
guaagacuug agaugaucct t 21

Claims (10)

1. the method for the lipid particle containing nucleic acid is manufactured, including：
(a) the 1st conductance for including nucleic acid in the 1st solvent is entered into microfluidic device；Wherein device has suitable for by one or more Multithread leads the 1st area of inflow device and for the 2nd area of the content that one or more streams are mixed with microfluid mixer；
(b) the 2nd comprising lipid particle-forming material in the 2nd solvent is flowed into gatherer to flow down in laminar flow condition to provide Dynamic the 1st and the 2nd stream, wherein lipid particle-forming material includes cation lipid, and wherein the 1st and the 2nd solvent is differed；
(c) one or more 1st streams and one or more 2 streams are made from the 2nd area of the 1st area's inflow device of device；And
(d) one or more 1st streams are mixed in the 2nd area of device and one or more 2 streams include lipid particle to provide The 3rd stream, the lipid particle includes：
(i) core being made up of nucleic acid and cation lipid；With
(ii) the 2nd lipid of the core is surrounded；
Wherein due to the nucleic acid, the 31P nuclear-magnetisms of the lipid particle measured in the solution after being handled with 150mM ammonium acetates Resonance is not presented for resonance spectrum.
2. the method for claim 1 wherein the nucleic acid is wrapped in lipid particle with 90~100% efficiency.
3. the method for claim 1 wherein the 1st solvent is aqueous buffer and the 2nd solvent is aqueous alcohol.
4. the method for claim 1 wherein the 2nd stream also includes the 2nd lipid.
5. the method for claim 1 wherein the 1st solvent is aqueous solvent and the 2nd solvent is organic solvent, and the wherein the 1st Volume ratio between solvent and the 2nd solvent is more than 1.0 (aqueous solvents:Organic solvent).
6. the method for claim 1 wherein the volume ratio is 3:1.
7. the method for claim 1 wherein the particle includes 30~95 molar percentage cation lipids.
8. the method for claim 1 wherein the 2nd lipid is selected from：Neutral lipid, anion lipid, and PEG- lipids.
9. the method for claim 8, wherein the 2nd lipid is selected from：PEG- lipids, amphion lipid, and sterol；Preferably wherein 2 lipids are selected from PEG- lipids and wherein described PEG- lipids are present in the liposome with the amount of 1.0~10 molar percentages In son.
10. the method for claim 1 wherein the particle has 15nm to 300nm diameter.
CN201710181826.6A 2009-11-04 2010-11-04 Lipid particle and related methods containing nucleic acid Pending CN107028886A (en)

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